POST-SYNTHETIC MODIFICATION OF POLYNUCLEOTIDES VIA ACYLATION REAGENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This- application claims benefit of U.S. Provisional Patent Application Serial No. 63/415,682, filed October 13, 2022, the disclosure of which is incorporated herein by reference.

BACKGROUND

[0002] The following information is provided to assist the reader in understanding technologies disclosed below and the environment in which such technologies may typically be used. The terms used herein are not intended to be limited to any particular narrow interpretation unless clearly stated otherwise in this document. References set forth herein may facilitate understanding of the technologies or the background thereof. The disclosure of all references cited herein are incorporated by reference.

[0003] Nucleic acid-polymer hybrids have emerged as a new class of biomaterials. They combine programmable self-assembly and biocompatibility with the versatility and diversity of synthetic polymers bringing enhanced properties of the molecular chimeras. Recent insights gained from the engineering of DNA -based materials and DNA-polymer conjugates have been instrumental in guiding the development of RNA-based nanomaterials and RNA- polymer hybrids.

[0004] The fabrication of the RNA-synthetic polymer hybrid materials has been facilitated through one of the following approaches: (1) noncovalent attachment of pre-synthesized polymers with RN A by electrostatic interactions or hydrogen bonding; (2) covalent grafting of pre~synthesized polymers onto RNA equipped with reactive handles through coupling reactions such as (strain-promoted or copper-catalyzed) azide-alkyne cycloadditions, amidation or disulfide exchange; (3) covalent conjugation of a polymerization initiator or acrylate moiety into RNA structures and subsequent polymerization from the RNA macroinitiators or grafting through RNA-acrylate macromonomers.

[0005] The noncovalent and the covalent coupling strategies have distinct advantages and disadvantages. For example, the noncovalent approaches may be applied to a broad range of RNA substrates regardl es s of the length , source , or structure of the RNA substrate for coupling. However, the weak and non-covalent interaction between nucleic acids and polymers often raises concerns about the stability of the complex as well as difficulties in control over the number- and binding site of polymers. In contrast, because of the specificity of the coupling chemistries, covalent modification methods allow for chemically stable and precise incorporation of pre-synthesized polymers, acrylate groups, or polymerization-initiating moieties into the predetermined sites in nucleic acids. However, coupling handles (for example, alkyne, thiol, norbornene, amine, etc.) must be incorporated into the nucleic acid through the solid-phase sy nthesi s or the use of an enzyme, which l imits the scope of the substrate and thus hinders practical and versatile application.

In other reactions involving RNA, acylating reagents have been studied as structural mapping agents for RNA via selective 2’-hydroxyl acylation analyzed by primer extension (sometimes referred to as SHAPE). SHAPE reagents have allowed RN A mapping even l» vitro and m vivo.

[0007] Among the various sources of nucleic acids, biomass extracts are receiving growing attention as building blocks, particularly for the synthesis of macroscopic structures. That attention is mainly due io the relati vely low cost of biomass DNA and RNA, compared to other types of nucleic acids. Additionally, such inexhaustible natural biopolymers can be extracted from any living organisms, potentially reducing the use of petrochemicals. For example, since there are approximately 50 billion metric tons of biomass DNA on the Earth, replacing the current production of commodity plastics would require converting only ca. 0.7% of the biomass DNA on the Earth. Consequently, a variety of macroscopic 3D structures, such as commodity plastics, hydrogels, aerogels, and optoelectronic devices have employed biomass DNA as the building blocks,

[0008| I hiiil recently, direct crosslinking of biomass DNA has been the main approach for fabricating biomass DNA-based material. Various non-covalent methods offer the advantage of reshaping and reprocessing the resulting material due to the weak and reversible nature of the interactions. The simple crosslinking of biomass DNA constrains the possibility of engineering material properties since incorporating functional molecules (for example, synthetic polymers) is challenging. It is not believed that the use of biomass RNA as building blocks in nucleic-acid based materials has been reported. [0009] It remains desirable to develop improved methods for the modification of nucleic acids, and particularly RNA with polymers.

SUMMARY

[0010] In one aspect, a method of modifying a polynucleotide includes reacting the polynucleotide with one or more molecules of an acylating reagent. The acylating reagent includes an acylating compound conjugated to (i) an initiator compound for reversible deactivation radical polymerization; (ii) a chain transfer agent for reversible deactivation radical polymerization or (iii) a compound comprising a polymerizable group to form a modified polynucleotide. The acylating reagent may have the formula:

R ₁ — C ₁ -( L ₁ )n-R ₂ wherein C ₁ is a spacer group, L ₁ is a linking group, wherein n is 0 or an integer in the range of 1 to 40, R ₁ is an active ester moiety, and Ra is a residue of the initiator compound for reversible deactivation radical polymerization, a residue of the chain transfer agent for reversible deactivation radical polymerization, or a residue of the compound comprising a polymerizable group. In a number of embodiments, n is iu the range of 0 to 20,

[0011] In a number of embodiments, C ₁ is selected from the group consisting of; wherein T is selected from the group O, S, -C(O)NH- or ~NHC(O)-, p is an integer in the range of 0- to 20 and q is an integer 1 to 20.

[0012] In a number of embodiments, L ₁ is selected from the group consisting of

wherein n2 is an integer in the range or 1 to 40, R’ is selected from the group of H, alkyl and aryl.

[0013] In a number of embodiments, R ₁ is wherein Ej, Ea, and E? are independently an electron donating group or an electron withdrawing group, X’ is F or CL E ₁ , E ₂ , and E ₃ may. for example, independently be H, Cl, F, Br or OH.

[0014] In a number of embodiments R ₁ has the formula: and E ₁ , E ₂ , and E ₃ are independently H, Cl, F, Br or OH.

[0015] to a number of embodiments, R2 is the residue for the initiator for reversible deactivation radical polymerization or the residue for the chain transfer agent for reversible deactivation radical polymerization and has the formula: wherein X is a homolytically cleavable group or a group activated by degenerative radical exchange;

Ra and R< are each independently selected from the group consisting of a homolytically cleavable group, a group activated by degenerative radical exchange, H, C1-C20 alkyl, C3-C8 cycloalkyl, C(-Y)R ₅ , C(=Y)NR ₆ R ₇ , COCl OH, CN, C2-C20 alkenyl, C2-C20 alkynyl, oxiranyl, glycidyl, aryl, heteroeyclyL aralkyl, aralkenyL C1-C6 alkyl in which from I to all of the hydrogen atoms are replaced with halogen and C1-C6 alkyl substituted with from I to 3 substituents selected from the group consisting of C1-C4 alkoxy, aryl, heterocyclyi, C(=Y)R ₅ , C(=Y)NR ₆ R ₇ , oxiranyl and glycidyl, and wherein R ₅ is C1-C20 alkyl, C1-C20 alkoxy, aryloxy or heterocyclyloxy, and Re and R? are independently H, or C1-C20 alkyl, or R6 and R7 rnay be joined together to form an alkylene group of from 2 to 5 carbon atoms, wherein Y is NR ₈ or 0 and R ₈ is H, straight, or branched C1-C20 alkyl or aryl.

[0016] In a. number of embodiments, X is selected from the group consisting of Cl, Br, I, nitroxyl, organotellurium, organostibine, organobismuthine, anti ~S- C(=S)-Z, wherein Z is selected from the group consisting of alkyl, alkoxy, alkylthio, aryl, and heteroaryl,

[0017] R ₃ , R ₄ may each independently be selected from the group consisting of Cl, Br, I, nitroxyl organotellurium, organostibine, organobismuthine, -S-C(=S)-Z, H, C1-C20 alkyl, C3- C8 cycloalkyl, C(-Y)R ₅ , C(=Y)NR ₆ R ₇ , COCI, OH, CN, C2-C20 alkenyl, C2-C20 alkynyl, oxiranyl, glycidyi, aryl, heterocyclyi, aralkyl, aralkenyl C1-C6 alkyl in which from 1 to all of the hydrogen atoms are replaced with halogen and C1-C6 alkyl substituted with from 1 to 3 substituents selected from the group consisting of C1-C4 alkoxy, aryl, heterocyclyi, C(=Y)R ₅ , C(=Y)NR6R7, oxiranyl and glycidyl. In a number of embodiments, R ₃ and R ₄ are each independently selected from the group consisting of H, C1-C20 alkyl, aryl and a heterocycle. R ₃ and R ₄ may each independently be selected fr om the group consisting of methyl, phenyl, pyridyl, substituted phenyl, substituted pyridyl and a heterocycle. In a number embodiments, X is selected from the group consisting of Cl, Br, I, -SC( = S)-Z, wherein Z is selected from the group consisting of alkyl, alkoxy, alkylthio, aryl, and heteroaryl. In a number of embodiments, X is selected from the group consisting of nitroxyl, -TeRy, -SbR ₉ R ₁₀ and -BiR ₉ R ₁₀ , wherein R» and R ₁₀ are each independently selected from the group consisting of aryl and a straight or branched C1-C20 alkyl group. In a number of embodiments, X is Br, R3 is methyl, R4 is methyl, and L ₁ is [0018] R2 may be a residue of a compound comprising a polymerizable group which is selected from the group consisting of a group including a vinyl group, a monocyclic alkene, and a bicyclic alkene. Rz may, for example, be selected from the group consisting of:

[0019] In a number of embodiments, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or hybrids thereof. The polynucleotide may, for example, be RNA or a hybrid including RNA. The RNA may be biomass RNA. The polynucleotide(for example, RNA) may be a single-strand polynucleotide. A degree of modi fication of RNA (or other polynucleotide) with the acylating reagent may be controlled by control of conditions of reaction of the RNA (or other polynucleotide) with the acylating reagent. The conditions of reaction may include one or more of the amount of acylating reagent, pH, and the ratio of an organic cosolvent to an aqueous phase used in the reaction. The cosolvent is a water-miscible, organic compound, which may be selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and acetonitrile (ACN), and acetone. In a number of embodiments, the cosolvent is DMSO. The conditions of reaction ( including one or more of the amount of acylating reagent, pH, and the ratio of the water-miscible, organic cosol vent to an aqueous phase used in the reaction) may be selected to promote reaction of the acylating reagent with nitrogenous bases of the polynucleotide.

[0020] In a number of embodiments, one or more positions of reaction of the acylating reagent on the polynucleotide (for example, RNA) is controlled by annealing at least one of one or more folly complimentary strands of a helper polynucleotide and one or more partially complimentary strands of a helper polynucleotide to a single strand of the polynucleotide before reaction of the acylating reagent with the polynucleotide. In the case of RN A, the helper polynucleotide is DNA in a number of embodiments hereof. The method may further include removing the at least one of the one or more fully complimentary strands of a helper polynucleotide and the one or more partially complimentary strands of a helper polynucleotide from the single strand of polynucleotide after reaction of the acylating reagent therewith. Removing the at least one of the one or more folly complimentary strands of a helper polynucleotide and the one or more partially complimentary strands of a helper polynucleotide from the single strand of polynucleotide may include selective degradation thereof.

[0021] In a number of embodiments, the acylating agent includes the residue of the initiator compound for reversible deactivation radica l poly merization or the residue of the chain transfer agent for reversible deactivation radical polymerization, and the method forther includes carrying out a reversible deactivation radical polymerization reaction with one or more monomers from the modified polynucleotide. The initiator compound for reversible deactivation radical polymerization may be an atom transfer radical polymerization (ATRP) initiator and the reversible deactivation radical polymerization may be an ATRP, In a number of embodiments, the ATRP is an oxygen-tolerant photoinduced ATRP reaction. The oxygen- tolerant photoinduced ATRP reaction may; for example, be mediated by eosin Y photocatalyst and a copper complex under green light irradiation. The one or more monomers may hydrophilic or hydrophobic.

[0022] In a number of embodiments, the acylating reagent includes an acylating compound conjugated to a residue of a polymerizable compound, wherein the polymerizable compound includes a vinyl group. The residue of the polymerizable compound is a moiety including a vinyl group, a monocyclic alkene, or a bicyclic alkene. The modified polynucleotide may be reacted as monomer in a polymerization reaction. The modified polynucleotide may be reacted as a crosslinking monomer. The modified polynucleotide may be reacted with one or more other monomers. In a number of embodiments; the polymerization reaction is a free radical polymerization or a reversible deactivation radical polymerization.

[0023] I n another aspect, a modified RNA compound is formed by reacting RNA with one or more molecules of an acylating reagent which is active to selectively acylate a 2’-hydroxyI group of the RNA, the acylating reagent includes an acylating compound conjugated to (i) an initiator compound for reversible deactivation radical polymerization; (ii) a chain transfer agent for reversible deactivation radical polymerization or (Hi) a compound comprising a polymerizable group to form a modified polynucleotide.

[0024] In another aspect, a compound has the formula: wherein CJ is a spacer group, La is a linking group, n is an integer in the range of 0 and 40, Eg Ea, and E? are independently an electron donating group or an electron withdrawing group, and Rs is residue of a polymerizable compound. Et, Ea, E?, C ₁ , L ₁ , and Rs may be as further described herein. In a number of embodiments, n is in the range of 0 to 20.

[0025] In another aspect, a modified polynucleotide composition includes a conjugate of a polynucleotide with one or more molecules of an acylating reagent. The acylating reagent includes an acylating compound conjugated to (i) an initiator compound for reversible deactivation radical polymerization; (ii) a chain transfer agent for reversible deactivation radical polymerization or (ii i) a compound comprising a polymerizable group. The acylating reagent is conjugated with the polynucleotide via reaction with at least one of: one or mote 2’-OH groups of the polynucleotide or one or more nitrogenous nucleobases of the polynucleotide.

[0026] In a further aspect, a polynucleotide-polymer hybrid composition is formed by performing a reversible deactivation radical polymerization reaction with one or more monomers from a modified polynucleotide composition. The modified polynucleotide composition includes a conjugate of a polynucleotide with one or more molecules of an acylating reagent. The acylating reagent includes an acylating compound conjugated to (i) an initiator compound for reversible deactivation radical polymerization; or (ii) a chain transfer agent for reversible deactivation radical polymerization. The acylating reagent is conjugated with the polynucleotide via reaction with at least one of: one or more 2'~OH groups of the polynucleotide or one or more nitrogenous nucleobases of the polynucleotide.

[0027] In still a further aspect, a polynucleotide-polymer hybrid composition is formed by performing a polymerization reaction with a modified polynucleotide composition. The modified polynucleotide composition includes a conjugate of a polynucleotide with one or more molecules of an acylating reagent. The acylating reagent includes an acylating compound conjugated to a polymerizable compound. The acylating reagent is conjugated with the polynucleotide via reaction with at least one of: one or more 2 ’-OH groups of the polynucleotide or one or more nitrogenous nucleobases of the polynucleotide. [0028] As used herein the terms “alkyl” (typically, C1-C20, that is from 1 to 20 carbon atoms), ’’alkenyl” (typically, C2-C20)” and “alkynyl” (typically, C2-C20) refer to straight- chain or branched groups (except for C1 and C2 groups). “Alkenyl” and “alkynyl” groups may have sites of unsaturation at any adjacent carbon atom position^) as long as the carbon atoms remain tetravalent, but, - or terminal (i,e., at the - and ( -Impositions) are present in a number of embodimen ts.

[0029] As used herein “aryl” refers to phenyl, naphthyl, phenanthryl, phenalenyl, anthracenyl, triphenylenyl, fluoranthenyl, pyrenyl, pentacenyl, chrysenyl, naphthacenyl, hexaphenyl, picenyl and perylenyl (preferably phenyl and naphthyl), in. which each hydrogen atom may be replaced with alkyl of from 1 to 20 c arbon atoms (preferably from 1 to 6 c arbon atoms and more preferably methyl), alkyl of from 1 to 20 carbon atoms (preferably from 1 to 6 carbon atoms and more preferably methyl) in which each of the hydrogen atoms is independently replaced by a halide (preferably a fluoride or a chloride), alkenyl of from 2 to 20 carbon atoms, alkynyl of from 1 to 20 carbon atoms, alkoxy of from I to 6 carbon atoms, alkylthio of from 1 to 6 carbon atoms, C3-C8 cycloalkyl, phenyl, halogen, NHa, Cl -C6- alkylamino, C1-C6-dialkylamino, and phenyl which may be substituted with from 1 to 5 halogen atoms and/or C1-C4 alkyl groups, (This definition of “aryl” also applies to the aryl groups in “aryloxy” and “aralkyl.”) Thus, phenyl may be substituted from 1 to 5 times and naphthyl may be substituted from 1 to 7 times (preferably, any aryl group, if substituted, is substituted from 1. to 3 times) with one of the above substituents. More preferably, “aryl” refers to phenyl, naphthyl, phenyl substituted from 1 to 5 times with fluorine or chlorine, and phenyl substituted from 1. to 3 times with a substituent selected from the group consisting of alkyl of from 1 to 6 carbon atoms, alkoxy of from I to 4 carbon atoms and phenyl. Most preferably, “aryl” refers to phenyl and tolyl.

[0030] In the context of the present invention, “heterocydyl” refers to pyridyl, furyl, pyrrolyl, thienyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyranyl, indolyl, isomdolyl, indazolyl, benzofuryl, isobenzofuryl, benzothienyl, isobenzothienyl, chromenyl, xanthenyL purinyl, pteridinyl, quinolyl, isoquinolyl, phthalazinyl, quinazolmyl, quinoxalinyl, naphthyridinyl, phenoxathiinyl. carbazolyl, cinnolinyl, phenamhridiuyl, acridinyl, 1,10- phenanthrolinyl, phenazinyl, phenoxazinyl, phenothiazinyl, oxazolyl. thiazolyl, isoxazolyl, isothiazolyl, and hydrogenated forms thereof known to those in the art. Preferred heterocyclyl groups include pyridyl, furyl, pyrrolyl, thienyl , imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl. pyridazlnyl, pyranyl and indolyl, the most preferred heterocyclyl group being pyridyl. Accordingly, suitable vinyl heterocycles to be used as a monomer in the present invention include 2 -vinyl pyridine,. 4-vinyl pyridine, 2-vi.nyi. pyrrole, 3-vinyl pyrrole, 2 -vinyl oxazole, 4- vinyl oxazole, 5-vinyl oxazole, 2-vinyl thiazole, 4-vinyl thiazole, 5-vinyl thiazole, 2-vinyl imidazole, 4-vinyl imidazole, 3-vinyl pyrazole, 4-vinyl pyrazole, 3-vinyl pyridazine, 4-vinyl pyridazine, 3-vinyl isoxazole, 3-vinyl isothiazoles, 2-vinyl pyrimidine, 4-vinyl pyrimidine, 5- vhiyl pyrimidine, and any vinyl pyrazine, the most preferred being 2-vinyl pyridine. The vinyl heterocycles mentioned above may bear one or more (preferably I or 2) C 1 -C6 alkyl or alkoxy groups, cyano groups, ester groups or halogen atoms, either on the vinyl group or the heterocyclyl group, but preferably on the heterocyclyl group. Further, those vinyl heterocycles which, when unsubstituted, contain an N--H group may be protected at that position with a conventional blocking or protecting group, such as a Cl -C6 alkyl group, a tris- Cl -C6 alkylsilyl group, an acyl group of the formula R ^W CO (where R ¹⁰ is alkyl of from 1 to 20 carbon atoms, in which each of the hydrogen atoms may be independently replaced by halide, preferably fl uoride or chloride),, alkeny l of from 2 to 20 carbon atoms (preferably vinyl), alkynyl of from 2 to 10 carbon atoms (preferably acetylenyl), phenyl which may be substituted w'ith from 1 to 5 halogen atoms or alkyl groups of from 1 to 4 carbon, atoms, or aralkyl (ary! -substituted alkyl, in which the aryl group is phenyl or substituted phenyl and the alkyl group is from 1 to 6 carbon atoms), etc. (This definition of “heterocyclyl” also applies to the heterocyclyl groups in “heterocyclyloxy” and “heterocyclic ring.”)

[0031] In general, any radically polymerizable alkene and cycloalkene can, for example, serve as a monomer for a polymerization reaction hereof or as a source for a residue of polymerizable compound in an acylating reagent hereof In a number of embodiments, monomers suitable for polymerization hi the presen t method include those of the formula: wherein R ^a and R ^b are independently selected from the group consisting of H, halogen, CN, straight or branched alkyl of from 1 to 20 carbon atoms (in a number of embodiments, from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms) which may be substituted with from 1 to (2n+1) halogen atoms where n is the number of carbon atoms of the alky! group (e.g. CFa), unsaturated straight or branched alkenyl or alkynyl of 2 to 10 carbon atoms (in a number of embodiments, from 2 to 6 carbon atoms, or from 2 to 4 carbon atoms) which may be substituted with from I to (2ml) halogen atoms (in a number of embodiments, chlorine) where n is the number of carbon atoms of the alkyl group (e.g. CH ² -CC 1 -), C3-C8 cyc loalkyl which may be substituted with from 1 to (2n- l) halogen atoms (preferably chlorine) where n is the number of carbon atoms of the cycloalkyl group, C(-Y)R ^e , C(-Y)NR ^f R ^g , YC(-Y)R ^e , SOR ^e , SCO ₂ R ^e , OSO ₂ R ^e NR ^h SO ₂ R ^e , PR ^e ₂ P(=Y)R ^e ₂ YPR ^e ₂ YP(-Y)R ^e ₂ NR ^h ₂ which may be quatemized with an additional R ^h group, aryl and heterocyclyh where Y may be NR ⁵ ’ S or O (preferably O); R ^e is alkyl of from 1 to 20 carbon atoms, alkylthio of from 1 to 20 carbon atoms, OR* (where R ^J is H or an alkali metal), alkoxy of from 1 to 20 carbon atoms, aryloxy or heterocyclyloxy; R ^f and R ⁸ are independently H or alkyl of from 1 to 20 carbon atoms, or R ^j and R ^g may be joined together to form an alkylene group of from 2 to 7 (preferably 2 to 5) carbon atoms, thus forming a 3- to 8-membered (preferably 3- to 6-membered) ring, and R ^h is IL straight or branched Ct- C ₂₀ alkyl or aryl;

R ^c and R ^d are independently selected from the group consisting of H, halogen (preferably fluorine or chlorine), C1-C6 (preferably C1) alkyl and COOR ^j (where R ^j is H, an alkali metal, or a C1-C6 alkyl group), or

R. ^a and R ^e may be joined to form a group of the formula (CH ₂ ), (which may be substituted with from 1 to 2n’, halogen atoms or C1-C4 alkyl groups) or C(=O)-Y-C(=O) ₅ where n' is from 2 to 6 (in a number of embodiments 3 or 4) and Y is as defined above. In a number of embodiments, at least two of R ^a , R ^b , R ^c and R ^d are H or halogen,

[0032] The present devices, systems, and methods, along with the attributes and attendant advantages thereof will best be appreciated and understood in view of the following detailed, description taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1A illustrates a representative scheme for synthesis of an atom transfer radical polymerization (ATRP) initiator-iunctionalized acyl imidazole reagent Br-Ala-AI for RNA modification.

[0034] FIG, IB illustrates an embodiment of a scheme for RNA functionalization with the Br- Ala-AI for the fabrication of RNA initiator. [0035] FIG. 1C illustrates a plot of hydrolysis kinetics of Br-AIa-AI reagent under the acylation condition (20% v/v DMSO in water).

[00361 FIG, ID illustrates a generalized scheme for modification of polynucleotides via reaction with acylating reagents hereof

[0037) FIG. 2 A illustrates schematically Br-Ala-AI treatment to RNA21 and DNA21 and polymerization from the oligonucleotides in the synthesis of 2’OH-selective modification using Br-AIa-AI.

[0038] FIG. 2B illustrates MALDI-TOF spectrum of RNA21 before Br-Ala-Al treatment.

[0039] FIG. 2C illustrates MALDI-TOF spectrum of RNA21 after Br-AIa-AI treatment ([M+H] ⁺ region expanded for clarity), wherein Y-axis is intensity (arbitrary unit).

[0040] FIG. 2D illustrates a SEC-MALS trace ofpOEOMA$o»-graftedRNA21 (solid line) and RNA21 initiator (dotted line), respectively.

[0041] FIG, 2E illustrates a MALDI-TOF of DNA21 before Br-Ala-Al reagent treatment.

[0042] FIG. 2F illustrates a MALDI-TOF of DNA21 after Br-AIa-AI reagent treatment.

[0043] FIG. 2G illustrates an SEC-MALS trace of pOEOMAsw-grafted treated DNA21 (solid line) and DNA21 initiator (dotted line), wherein reaction conditions for FIGS, 2 A through 2G were: [OEOMA ₅₀₀ ] - 300 mM, [EYHs] - 0.015 mM, [CuBr ₂ ] - 0.9 mM, [TPMA] - 2.7 mM, [RNA21 or DNA21] - 0.075 mM under the green LEDs (520 nm, 3.7 mW /cm ² ) for 30 min at r,t., in PBS, the monomer conversion was determined by Til NiMR spectroscopy, and PBS was used as an eluent for SEC-M ALS analysis.

[0044] FIG. 3A illustrates an embodiment of a scheme of site-selective acylation on the RNA substrate ( RNA21) within DNA duplex by using fully complementary helper DNA (fcDNA21) or partially complementary helper DNA with a mismatch in the middle (pcDNA21).

[0045] FIG. 3B illustrates MALDI-TOF spectra after Br-AIa-AI -treated RNA21 protected by fcDNA21

[0046] FIG. 3C illustrates MALDI-TOF spectra after Br-AIa-AI-treated RNA21 protected bypcDNA21 [0047] FIG.3B illustrates an SEC-MALS trace of pOEOMA ₅₀₀ --grafted RNA21 (solid line) prepared by acylation at induced mismatch using pcDNA21 as the helper DNA, wherein the green doted line is a trace of the RNA21 initiator, and general polymerization, conditions identical to those set forth in the description of FIG. 2G above were employed ([RNA oligo] = 0.075 mM (i.e., ca. 0.1125 m.M of alkyl bromide).

[0048] FIG. 3E illustrates a scheme for RNA32 functionalization with Br-AIa~AI in a hairpin RNA modification with 2 -OH protection using fully-complementary DNA (fcDNA32).

[0049] FIG. 3F illustrates a scheme for RNA32 functionalization with Br-AIa-AI in a hairpin RNA modification without 2'-OH protection using fully-complementary DNA (fcDNA32).

[0050] FIG. 3G il lustrates a MALDI-TOF spectrum of untreated hairpin RNA.

[0051] FIG. 3H illustrates a MALDI-TOF spectrum of Br-Ala-Al-treated hairpin RNA.

[0052] FIG. 31 illustrates a MALDI-TOF spectrum of Br-AIa-AI-treated hairpin RNA protected by/cDNA32 prior to treatment, wherein 32mer hairpin RNA (RNA32) was used as a substrate, the single-stranded loop in the RNA32 is 4 nucleotides long flanked by 2 GU wobble pairs, the acylation efficiency for RNA32 was slightly higher (approximately 65%) than for RNA21 (approximately 30%), and this difference in the efficiency may, for example, be attributed to the difference in the number of accessible 2 -OH groups present in the respective RNA sequences which affects the ratio of acylating reagent to 2 -OH groups: ca. 143:1 for RNA21 ; and m. 391:1 for RNA32.

[0053] FIG. 4A illustrates a schematic representation of an embodiment of a water-free functionalization process in 100% DMS0.

[0054] FIG. 4B illustrates UV-Vis spectra RNA21. after Br-Ala-AI treatment under different reaction conditions.

[0055] FIG. 4C illustrates a MALDI-TOF spectrum of RNA21 substrate after water-free Br- Ala-AI treatment for 4 h.

[0056] FIG. 4D illustrates a MALDI-TOF spectrum of RNA21 substrate after water-free Br-

Ala-AI treatment for 24 h. [0057] FIG. 5A illustrates a scheme of biomass R.NA initiator preparation and grafting from biomass RNA initiator.

[0058] FIG, SB illustrates a photograph of biomass RNA after overnight incubation in anhydrous DMSO (left) without; and (right) with Br-Ala-Al reagent, respectively.

[0059] FIG. 5C illustrates UV-Vis spectra of biomass RN A after Br-Ala-AI treatment under different reaction conditions.

[0060] FIG. 5» illustrates a molecular weight control experiment using Br-Ala-A I-treated biomass RNA the as the initiator wherein the result of the polymer characterization by SEC- MAIN is shown in Table 1 below.

[0061] FIG. 5E illustrates relative fluorescence intensity of pOEOMA ₅₀₀ -grafted biomass RNA after staining with SYBR Gold, (ex: 495 nm, cm: 537 nm).

[0062] FIG, 5F illustrates a chain extension experiment using biomass RNA initiator, wherein all polymerizations were conducted under the general polymerization condition using OEOMA ₅₀₀ as the model monomer in PBS under green light irradiation for 30 min.

[0063] FIG 6A illustrates a scheme for bmRNA-pOEOMA hydrogel synthesis by copolymerization of OEOMA ₅₀₀ and PEGDMA ₇₅₀ from AmRNA macroinitiator.

[0064] FIG. 6B illustrates a photographic image of ZraRNA-pOEOMA hydrogel after 30 min of polymerization using NnRN A initiator (final concentration of 0.5 mg/mL) under the general polymerization condition in the presence of PEGDMArso (final concentration of 60 mM), wherein (left) ZwRNA macroinitiator synthesized under water-free conditions was used as the initiator, and (right) unmodified biomass RN A without Br-AIa-AI treatment was used, and wherein a light pink color was observed due to eosin Y photocatalyst

[0065] FIG. 6C illustrates photographic images after polymerization of NIP AM with biomass RNA (left) with; and (right) without Br-Ala-AI treatment, respectively, wherein the reaction conditions were: [NIPAM]/[EYH2]/[CuBr2]/[Me6TREN] = 1000/0.045/2.7/8.1 , [bmRNA] - 0.5 mg/mL and [NIPAM] = 1.000 mM under green light irradiation (520 nm, 3.7 mW cm ^-2 ) for 30 min, in 80% v/v DMSO in PBS. MeeTREN = tris[2-(dimethylamino)ethyl]amme. [0066] FIG. 7A illustrates, synthesis of the AAm-AI reagent and the treatment of biomass RNA with AAm-AI under different conditions to engineer the degree of modification.

[0067] FIG, 7B illustrates the number of acrylamide modifications per 10 ribonucleotides. IX and 3X AAm-AI refer to I and 3 equivalents of AAm-AI compared to ribonucleotides, respectively wherein ¹ H NMR was used for the quantification.

[0068] FIG. 7G illustrates hydrolysis kinetics of AAm-AI reagent under the different concentrations of DM SO in water based on NMR peak analysis.

[0069] FIG. 7D illustrates UV-Vis spectra of acrylamido RNA cross l inker synthesized under different conditions.

[0070] FIG. 8 illustrates the effect of the degree of modification to the degradability of RNA hydrogels in 15% FBS, wherein the 100R ₁₀₀ , 50R ₁₀₀ , and 25R ₁₀₀ hydrogels were synthesized by homopolymerization of RNA crosslinker that was prepared under the 100%. 50%, and 25% of DMSO in water (Wv), respectively, and he RNA hydrogels were stained with GelRed dye for easier visualization. Scale bars = 5 mm.

[0071] FIG. 9 A illustrates a scheme of copolymerization of RNA crosslinker in water or DMSO.

[0072] FIG. 9B illustrates synthesis of RN A-NIPAM hybrid gels and staining with GelRed. (1—4) NIPAM gel without RNA (1), 25R _5S NIPAM ₄₅ (2), 5ORS5NIPAM45 (3), and 100R ₅₅ NIPAM ₄₅ (4), respectively.

[0073] FIG. 9C illustrates RNA-acrylamide hybrid gel (25R«7AAmix.w) synthesized in the custom-designed mold.

[0074] FIG. 9D illustrates comparison of compression modulus of RNA-acrylamide hybrid gels with 10, 30, or 90 mg/mL of RN A cross linkers synthesized under different acylation conditions (25% or 100%).

[0075] FIG, 9E illustrates synthesis of RNA-MA hybrid gel in DMSO. MA with Irgacure 2959 (left), M A and unmodified RN A with Irgacure 2959 (middle), and MA and RNA X-linker with Irgacure 2959 (right. lOORszMAss). Scale bars = 1 cm. [0076] FIG. 9F illustrates a contact angle test of RNA hydrogels with different surface polarities.

[0077] FIG, 10A illustrates a scheme of methacrylic RNA crosslinker and copolymerization by different radical polymerization methods showing reaction of biomass RNA with HEMA- CM reagent and subsequent copolymerization with OEOMA ₅₀₀ via EY/Cu-mediated photo* ATRP or PET-RAFT (reversible addition fragmentation chain-transfer polymerization) under green light, irradiation (λ = 540 nm).

[0078] FIG. 10B illustrates a digital camera image of RNA-OEOMA ₅₀₀ hybrid hydrogels fabricated by PET-RAFT.

[0079] FIG. 10C illustrates a digital camera image of RNA-OEOMA ₅₀₀ hybrid hydrogels fabricated by photo-ATRP.

[0080] FIG. 10D illustrates the swelling ratio of RNA-OEOMAsw hybrid hydrogels made by PEI-RAFI; FRP, and photo-ATRP, wherein the standard deviation was calculated from 3 different batches. Abbreviations'. TPM A (tris(2-pyridylmethyl)amine); HEBiB (2- Hydroxyethyl 2-bromoisobutyrate); TEOA (triethanolamine); CPADB ( 4-Cyano-4- (phenylcarbonothioy lthio)pen tanoic acid) .

[0081] FIG. 11 A schematically illustrates silver-doping as the route to grant electrical conductivity to the RNA hydrogel showing the binding, nucleation, and RNA-templated growth of Ag in the RN A hydrogel.

[0082] FIG. 1 IB illustrates a digital camera image of AgN On-treated polyacrylamide gels with or without RNA. (1 -3) 50R50AAmix50 after the treatment with () (1), 100 (2 k and 400 mM (3) of AgNOs for 3 h, respectively. (4 -6 ) Polyacrylamide gels after the treatment with 0 (4), 100 (5), and 400 mM (6) of AgNCh for 3 h, respectively.

[0083] FIG. 11C ill ustrates SEM and EDX element mapping of RNA-acrylamide hydrogel. Top lane: RNA-acrylamide hydrogel (2 in FIG. 11B). Bottom lane: polyacrylamide hydrogel (5 in FIG. 11B). The inset in the SEM image at the leftmost is the S EM image of the hydrogel before cross-sectional cutting. Scale bars = 500 pm.

[0084] FIG. 1 ID illustrates electrical conductivity measurement of RNA-acrylamide hydrogels with different RNA content (0-50 wt%) after overnight treatment with 100 mM of AgNO3. (E) Digital camera image of the AgNffo-treated RNA hydrogels used for the electrical conductivity test. From left, polyacrylamide gel, 50R6AAmix94, 50R11AAmix89, 50R20AAmix80, 50r33AAmix67, and 50R50AAmix50, respectively.

DETAILED DESCRIPTION

[0085] It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described representative embodiments. Thus, the following more detailed description of the representative embodiments, as illustrated in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely il lustrative of representative embodiments.

[0086] Reference throughout this specification to “one embodiment” or “an embodiment” (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” or the like in various places throughout this specification are not necessarily ah referring to the same embodiment,

[0087] Furthermore, described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments. One skilled in the relevant art will recognize,, however,, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, et cetera. In other instances, well known structures, materials, or operations are not shown or described in detail to avoid obfuscation.

[0088] As used herein and in the appended claims, the singular forms “a,” “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a reagent” includes a plurality of such reagents and equivalen ts thereof known to those skilled in the art, and so forth, and reference to ‘-the reagent” is a reference to one or more such reagents and equivalents thereof known to those skilled i n the art, and so forth. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value, as well as intermediate ranges, are incorporated into the specification as if individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contraindicated by the text.

[0089] In a number of representative embodiments hereof, strategies for the preparation of modified oligonucleotides or polynucleotides such as polynucleotide-polymer hybrids are set forth. Modified polynuc leotides hereof may inc lude one or more incorporated initiators, one or more incorporated chain transfer agents, or one or more incorporated polymerizable moieties or handles. Compositions hereof further include polymers formed from such modified polynucleotides. Examples of suitable polynucleotides include, but are not limited to, polynucleotides and oligonucleotide sequences, including DNN, RNA, DNA/RNA hybrids, peptide nucleic acids (E’NA), locked nucleic acid (LNA), and derivative or analogs thereof, which may be double stranded or single stranded and include, without limitation., synthetic polynucleotides that may be administrated to a patient.

[0090] The term “polymer” or the prefix “poly” (when referring to a particular type of polymer such as a polynucleotide) refers generally to a molecule, the structure of which includes repeat units derived, actually or conceptually, from molecules of low relative molecular mass (monomers). The term “oligomer” or the prefix “oligo” (when referring to a particular type of oligomer such as an oligonucleotide) refers generally to a molecule of intermediate relative molecular mass, the structure of which includes a small plurality of units derived, actually or conceptually, from molecules of lower relative molecular mass (monomers). In general, a. polymer is a compound having >1 , and more typically >13 repeat units or monomer units, while an oligomer is a compound having >1 and <20, and more typically less than 13 repeat units or monomer units. The term polymer thus includes oligomers as well as molecules of higher molecular weight.

[0091] hi a number of representative embodiments, synthetic methods hereof achieve both (i) direct and (ii) covalent modification of a polynucleic acid or polynucleotide (such as DNA, RNA, PNA, LNA or hybrids of polynucleotides, including homo or heterogenous structures) or a derivative or analog thereof, while reducing or eliminating many of the problems related to current synthetic methods. Further, the synthetic methods hereof expand the scope of polynucleotide substrates from short synthetic oligonucleotides to longer transcripts or biomass nucleic acids (that is, biomass RNA (bmRNA) and biomass DNA (bmDNA)) extracted from natural sources. To facilitate covalent polynucleotide modification in a universal and direct manner, acylating reagent chemistry, as exemplified by representative acyl imidazole chemistry, was used. Acyl imidazole chemistry includes the activation of a carboxylic acid with an imidazole leaving group, which could selective covalently react with 2’-hydroxyl (2 - OH) groups in RNA to form 2‘-O-adducts (see FIGS. 1A through 1C). Acylating reagents such as acyl imidazole reagents may also react with nitrogenous nucleobases. Reaction of acylating reagents with nucleobases may be promoted via control of reaction conditions (for example, control of water, aqueous content ).

[0092] Compared to other reagents that, are known to react with nucleic acids (for example, dimethyl sulfate, ninhydrin, psoralen, diazo compounds, etc,), acylation reagents, and particularly acyl imidazole reagents, have significant advantages, such as high reaction yield and site-selective incorporation, while generating a less toxic and biocompatible by-product (for example, imidazole). As used herein, acylating reagent are organic reagents via which functional groups may be added to RNA via an acyl group ( R-C(=O)-R’). Acylation may, for example, resul t in the formation of an am ide or an ester linkage.

[0093] In a number of studied embodiments hereof one or more molecules of an acylating reagent hereof were reacted with a polynucleotide. The acylating reagent includes an acylating compound conjugated to at least one of: a reversible deactivation radical polymerization initiator compound, a chain transfer agent for eversible deactivation radical polymerization or a compound comprising a polymerizable group. In the case that the acylating compound is conjugated to a polymerizable group, the resulting modified polynucleotide may be reacted as a monomer or a crosslinking agent. The acylating reagent may be active to selectively acylate a 2 -hydroxyl group, which is present in RNA. However, acylating reagents hereof may also be acti ve to react with nucleobases present in polynucleotides.

[0094] In a number of studies, reversible deactivation radical polymerization (RDRP) methods were utilized for the preparation of modified polynucleotides or polymicleotide conjugates. In general, RDRP procedures exhibit tolerance towards functional monomers and functional groups present, in nucleic acids and drugs. The three most common RDRP methods are atom transfer radical polymerization (ATRP), nitroxide mediated polymerization (NMP) and reversible addition fragmentation chain-transfer (RAFT) systems, each of allows unprecedented control over polymer properties such as dimensions (molecular weight), uniformity (polydispersity), topology (geometry), composition and functionality. See, for example, Matyjaszewski, K., Davis, T. P,, Eds. Handbook of Radical Polymerization; Wiley: Hoboken, 2002 chapter 11 pp 523-628; Matyjaszewski, K.; Xia, J. Chem. ftev. 2001, 101, 2921- 2990; Braunecker, W. A.; Matyjaszewski, K. Progress in Polymer Science 2007, 32, 93-146; Siegwart, D. L; Oh, 1 K.; Matyjaszewski, K. Prog. Polym. Sei. 2012, 37, 18-37; Hawker, C. L; Bosman, A. W.; Harth, E. Chemical Reviews 2001 , 101, 3661-3688; Mead, G.; Rizzardo, E,; Thang, S. H. Aust. J, Chem. 2012, 65, 985-1076, the disclosures of which are incorporated by reference. Degenerative transfer procedures have also been utilized as discussed in Tasdelen, M.A. et al., Telechelic Polymers by Living and ControlledZLiving Polymerization Methods, Brog. Polyrn. Set. 2011 , 5(5, 455-567.

[0095] ATRP is one of the most widely used controlled radical polymerization techniques for the synthesis of polymer-biohybrids. See, for example, Matyjaszewski, K.; Tsarevsky, N. V., Macromolecular Engineering by Atom Transfer Radical Polymerization. <Z Am. Chem. See. 2014, 136 (Copyright (C) 2014 American Chemical Society (ACS). All Rights Reserved.), 6513-6533, Wang, J.-S.; Matyjaszewski, K., Controlled/Giving" radical polymerization, atom transfer radical polymerization in the presence of transition-metal complexes. J. Am. Chem. Sac. 1995, 117 (20), 5614-15 and Averick, S.; Mehl, R. A.; Das, S. R.; Matyjaszewski, K., Well-defined biohybrids using reversible-deactivation radical polymerization procedures. J. (.'onirohW /teZease 2015, 205, 45-57, and Lorandi, F.; Fantin, M.; Matyjaszewski, K., Atom Transfer Radical Polymerization: A Mechanistic Perspective. J. Am. Chem. Sac. 2022, 144 (34), 15413-15430, the disclosure of which are incorporated herein by reference. In ATRP, polymerization is initiated from an alkyl halide initiator (R-X, where X can be either Cl or Br) through the reversible activation and deactivation cycles, typically mediated by a copper catalyst. See, Truong, N. R; Jones, G. R.; Bradford, K. G.; Konkolewicz, D.; Anastasaki, A., A comparison of RAFT and ATRP methods for controlled radical polymerization. AGz. Rev. Chem. 202.1, 5 (12), 859-869, Matyjaszewski, K.; Xia, J., Atom Transfer Radical Polymerization. Chem. Z?ev. 2001, 101 (9), 2921-2990, the disclosures of which are incorporated herein by reference. In a number of embodiments hereof, an ATRP initiator may be incorporated in a polynucleotide such as RNA and a polymer grafted from the polynucleotide rather than grafting a polymer onto pre-synthesized polymers. Such a ‘grafting- from’ approach may, for example, avoid potential steric hindrance and purification issues associated with the "grafting-outo’ approach. Initiating polymerization directly from the polynucleotide provides for easier purification of the final product (separation of high- molecular-weight polynucleotide-polymer hybrids from unreacted low-molecular-weight monomers), while expanding the range of architectural complexity by dramatically reducing steric hindrance. [0096] Among the various ATRP methods reported, an oxygen-tolerant photoinduced ATRP (photo-ATRP) technique mediated by eosin ¥ (EYFh) photocatalyst and a copper complex (X- Cu ⁿ /L) under green light .irradiation was used herein. See Szczepaniak, <3.; Jeong, J.; Kapil, K.; Dadashi-Silab, S.; Yemeni, S. S.; Ratajczyk, P.; Lathwal, S.; Schild, D. I; Das, S. R.; Matyjaszewski, K., Open-air green-light-driven ATRP enabled by dual pbotoredox/copper catalysis. Chem. 2022, 13 (39), 1 1540-1 1550 and Kapil, K.; Jazani, A. M.; Szczepaniak, G.; Murata, H.; Olszewski, M.; Matyjaszewski, K_, Fully Oxygen-Tolerant Visible-Light- Induced ATRP of Acrylates in Water: Toward Synthesis of Protein-Polymer Hybrids. Macro/wo/eca/a? 2023, 56 (5), 2017-2026, the disclosures of which are incorporated herein by reference. In that green-lighi-driven ATRP, the excited eosin Y photocatalyst transfers an electron to the X~Cu ^Ji ZL complex, thereby (regenerating the ATRP activator (Cu ^l /L) required for polymer growth. Compared to other ATRP methods, the photoredox/Cu-catalyzed A TRP method is particularly advantageous for polymerization from nucleic acid initiators as a result of the excellent oxygen tolerance of the dual catalysis, which allows for polymerization in low volumes (150--250 gL) without the need for deoxygenation processes such as Ni purging or freeze-pump-thaw cycles. Such processes are typically challenging for low-volume polymerization with limited amounts of initiators and may risk mechanical degradation of the nucleic acid.

[0097] In a number of representative studies hereof using representative acyl imidazole acylation chemistry, an ATRP initiator-functionalized acyl imidazole reagent enabled covalent modification of RNA as well as DNA in a post-synthetic (that, is, after solid-phase synthesis or after transcription) reaction. In a similar manner, an acylating reagent, may be conjugated to a chain transfer agent (for RAFT polymerization). Following the integration of the ATRP initiator or the chain transfer agent , a subsequent controlled polymerization process results in the controlled growth of polymer from the polynucleotide with, for example, a narrow molecular weight distribution. In further representative studies, a polymerizable-group- fonctionalized (for example, a viny l-group functionalized ) acyl imidazole reagent hereof, may be used to functionalize a polynucleotide with reactive/polymerizablc groups.

[0098] Unlike SHAPE reagents for 2 -OH modification used as probes in interrogation of RNA structure and function (for example, analogs of isatoic anhydride), acyl imidazole-based reagents hereof are particularly usefol for RNA modification because they often exhibit longer half-lives (over 30 min) with quantitative reaction yields. In a number of embodiments, a representative synthetic strategy hereof included a coupling reaction between 11’-carbonyldiimidazole (CDI) and p-alanine ATRP initiator (Br-Ala) to obtain an ATRP initiator-acyl imidazole reagent (FIG. 1A). In the general formula for acylating reagents set forth above, set forth above, R ₁ is an imidazole group, Ct is ~ -(CHsJs, L ₁ is ~NH-C(O)~, and Ra is -C(CHs)2Br. An amide-based ATRP initiator (FIG. 1A, Br-Ala) was chosen in representative examples because of the higher solubility of amides in aqueous buffers compared to ester-based ATRP initiators. The simple mixing of CDI with the Br-Ala in anhydrous DM SO successfully yielded the Br- Ala-functionalized acyl imidazole (Br-Ala-AI) reagent. The structure of the reagent was confirmed by NMR spectroscopy. The hydrolysis kinetics of Br-Ala-AI under coupling conditions (20% v/v DMSO in water) was also investigated to estimate the reactivity of the reagent and determine its half-life (FIG. 1C). The half-life of Br-Ala-Al was 38 min, indicating that the reactivity of Br-Ala-AI was comparable to previously reported acyl imidazole reagents.

[0099| To test the reactivity of Br-Ala-AI. a 21 -mer RNA (RNA21) and a 21 -met DNA (DNA2I ) of similar sequence (that is, including thymine instead of uracil; see Table 1 for sequences) were treated with Br-Ala-AI in 20% v/v DMSO in nuclease-free water (FIG. 2A). After 4 h of gentle shaking at room temperature, the RNA21 or D.NA21 was purified by isopropanol precipitation and three subsequent repeated centrifugation steps with a 3K molecular weight cut-off filter (MVVCO filter) to remove residual unreacted and hydrolyzed Br-Ala-AI. Finally, the RNA21 and DNA21 strands were analyzed by mass spectrometry to determine the number of ATRP initiators atached. The comparison of the mass spectra of RNA21 before (FIG. 2B) and after (FIG. 2C) the treatment of Br-Ala-AI revealed that the peak for the initial RNA sequence at 6648.2 m/z disappeared after the reaction, indicating efficient RNA functionalization. Additionally, the normal distribution of the masses of the reaction products suggested that an average of six ATRP initiators were atached to the RNA21 . In stark contrast, no dramatic change in the mass spectrum was observed when DNA2I was reacted with the Br-Ala-AI (FIGS. 2E and 2F). Although a small peak at 6603.8 m/z corresponding to 1 ATRP initiator attachment was observed, that observation was likely a result of the coupling to the hydroxyl group at either the 5' or 3' terminus. The experiments with RN A21 and DN A21 successfully demonstrated that the incorporation of ATRP initiators selectively onto 2 -OH groups in RNA is achievable. The ATRP initiators can be incorporated into post-synthetic RNA without, for example, relying on modifications added during the solidphase RNA synthesis. Table 1. Sequences of oligonucleotides used in this study.

[00100] The ability of the incorporated tertiary a-bromoisobutyramide moieties in RNA to initiate ATRP was also investigated. The Br-Ala-Al treated RNA21 initiator (FIG. 20) was used to perform the polymerization of OEOMA.w> (that is, oligof ethylene oxide) methyl ether methacrylate, average Ms = ^: 500) using EYEh as the photocatalyst and CuBn/TPMA (TPMA ~ tris(2-pyridylmedryi)amine) as the catalyst under the green light irradiation (520 nm) in phosphate-buffered saline (PBS) without deoxygenation. After the polymerization, the resulting RNA21«pOEOMA$oo was dialyzed three times using 100K MWCO filters, passed through a Sep-Pak Cl 8 reverse-phase cartridge to remove the photocatalyst and unreacted OEOMA ₅₀₀ , and analyzed by size-exclusion chromatography equipped with multi-angle light scattering detector (SEC-MALS). Despite the low RNA21 initiator concentration of 0.075 m.M , a relati vely high monomer conversion of 25% and a clear shift of the SEC-MALS trace to the high molecular weight area were observed ( FIG. 2D). This may be atributed to the multiple initiators in a single RNA molecule (alkyl bromide = ca. 0.45 mM). The disappearance of the initiator peak (elution volume of ca. 21.4 min) indicates a nearly quantitative initiation from the RNA21 initiator, which is also confirmed by the good agreement between the absolute molecular weight obtained from SEC-MALS and the theoretical molecular weight In contrast to the reaction with RNA21 initiator, no monomer conversion was observed, when the Br-Ala-AI-treated DNA21 was used as an initiator (FIG. 2G). The SEC- MALS peak did not shift, indicating the absence of any initiator functionalization of the DNA sequence. The radicals generated from the residual ATRP initiator may have been at too low concentration and were scavenged by traces of oxygen and other impurities. [00101] Another reagent using a-bromoisobutyric acid (BiBA-AI ) was synthesized and tested. The monomer conversion with BiBA-AI -treated RNA21 was not different from that of control experiments. This result is likely to be due to the rapid hydrolysis of the BiBA-AI reagent in aqueous media arising from the short distance between the strong electron withdrawing group (that is, Br) and acyl imidazole.

[00102] Control over the number and the position of polymer chains in nucleic acid- polymer hybrids (for example, multiblock copolymers, miktoarm stars, bottlebrushes, etc.) can provide a powerful approach to tailoring their properties. With appropriately positioned polymer chains, properties such as self-assembly, mechanical strength, and therapeutic efficiency can be controlled. Control over the amount of acylation was used to engineer the architecture of the hybrids. A helper polynucleotide such as helper DNA that hybridizes with an RNA substrate can sequester 2'-OHs from acylation while leaving the hydroxyl groups at the predetermined positions exposed and reacti ve.

[00103] Control over the incorporation of the ATRP initiator groups in RNA21 was studied, by introducing fully complementary polynucleotide (for example, representative DNA (fcDNA21)) or partially complementary polynucleotide (for example, representative DNA (pcDNA21)) prior to Br-AIa-AI treatment. Once annealed, the fcDNA21/RNA21 heteroduplex is completely hybridized with 2’-OH groups inaccessible to reactions, whereas the pcDNA21 sequence annealed to SR.NA21 , results in a single-nucleotide mismatch in the middle of the heteroduplex (FIG. 3A). It was hypothesized that 2‘-OH groups at the locally perturbed structure (that is, mismatch) would be more accessible to the Br-AIa-AI. The annealed heteroduplex was treated with the Br-AIa-AI according to the previously established procedure. The reaction was conducted in MOPS buffer (pH = 7,5) for 4 h. Selective DNA degradation was then performed using DNase I. The remaining RNA product was isolated by isopropanol precipitation and filtration using a 3K MWCO filter and characterized by mass spectrometry. A significantly lower number of modifications in RNA21 was observed for the fc DNA21/RNA2i duplex and most of the RNA21 remained unmodified (FIG. 3B). The low number of acylations in FIG. 3B is likely to occur at the terminal hydroxyl groups on the 5' and 3' ends of the RNA, In contrast, the use of poDNA21 as a helper DNA resulted in a moderate acylation yield in the range of 0 to 4 modifications (FIG. 3C), which is less than compared to the result with single-stranded RNA21 (FIG. 2C) and more than the result with RNA21-/bDNA21 duplex (FIG. 3B). It lias previously been shown that unpaired nucleotides. such as loops, bulges, mismatches, and nicks, can render adjacent nucleotides thermodynamically less stable and conformationally flexible. Consequently, acylation around the mismatch/unpaired site occurs in addition to the primary reaction she (that is, the mismatch in the present case). This can be observed through reverse transcription and subsequent polyacrylamide gel electrophoresis (PAGE).

[00104] Photoinduced ATRP was performed using the RN.A21 initiator prepared in the presence of pcDNA21 helper DNAs. As shown in FIG. 3D, a monomer conversion of 15% was observed, confirming successful initiation and polymerization. The conversion was lower than the previous result in FIG. 2D (25%) as a result of fewer initiators within a single RNA molecule. Notably, the MMAIS was higher than the theoretical molecular weight which may be a result of unreacted residual initiators (a peak at an elution volume of ca 21.4 min). The residual initiator peak originates either from unmodified RNAs (FIG. 3C) or from the lower initiation efficiency (trapping with impurities) at such a low initiator concentration.

[00105] A hairpin RNA (RNA32), which contains a double-stranded stem and a singlestranded loop in an individual RNA strand was also tested. The stem in the hairpin was able to sequester and protect some of the 2’-OHs in the RNA in a double-stranded region ( FIG. 3E through 31). Consistent with the results of the DNA-guided acylation, the clear shift of the mass peak of the hairpin RNA (FIGS, 3G and 3H) was inhibited when the hairpin RNA was protected by a fully complementary DNA sequence (/cDNA32, FIG. 31 ). These results indicate that control over the incorporation of ATRP initiators with selectivity within RNA sequences could be achieved by using an appropriate helper DNA, This strategy is useful to control the density of initiators in RNA sequences toward engineering new architectures in RNA-polymer hybrids (whether the acylation reaction occurs at the 2’-OHs and/or at the nitrogenous nucleobases).

[00106] With both moderate acylation on single-stranded RNA and controlled acylation reactions on R.NA/DNA. duplexes to incorporate the initiators within RNA sequences, the possibility of enhancing the initiator incorporation to reach a nearly quantitative yield (that is, 1 modification per nucleotide) was explored, which could, for example, be useful for RNA bottlebrush synthesis and engineering. Bottlebrush polymers are described, for example, in Xie, G.; Martinez, M. R.; Olszewski, M.; Slteiko, S. S.; Matyjaszewski, K., Molecular Bottlebrushes as Novel Materials. Zhrwrutorrurjo/ecu/es 2019, 20 f l), 27-54. Relatively high levels (25-50%) of 2 ’-OH ftrRctionalizatioR has been reported by improving the water solubility of the acylating reagents, tuning the reactivity of the acylating reagents, or using nucleophilic catalysts (for example, DMAP,4-dimethylaminopyridiiie). Nonetheless, the hydrolysis of the reagents under typical reaction conditions ( 10-30% v/v DMSO in aqueous buffer) could prevent reaching the stoichiometric level of reac tion tha t we desired.

[00107] To suppress the hydrolysis of Br-AIa-AI and accomplish quantitative functionalization, the reaction was carried out under water-free conditions (that is, 100% DMSO, anhydrous), as shown in FIG. 4A. For this water-free fiinctionalization reaction, RNA21 (20 nmol) was lyophilized to obtain a dry pellet and 100 pL of 0.6 M Br-AIa-AI in anhydrous DMSO was added to the container. Two different reaction times were tested and after 4 and 24 h of incubation with gentle shaking, the RNA products were isolated by isopropanol precipitation and subsequential filtration using a MWCO filter. A significant difference between the mass spectra taken after 4 h (FIG. 4C) and 24 h ( FIG. 41)) of the reaction was observed. First, the shape of the observed mass distribution was different. As seen in FIG. 4C, the peak from RNA21 at 6648.2 m/z remained as the largest peak and the intensity decreased progressively, which may be a result of the stepwise coupling of the Br-AIa-AI: one functionalization on the single RNA nucleotide enhances the solubility of the RNA strands in DMSO which allows and enhances the next coupling reaction. In contrast, after 24 h of reaction in DMSO (FIG. 41)), a normal distribution of mass peaks was observed, suggesting that the RNA strands were completely dissolved in DMSO and the Br-AIa-AI reacted with all the hydroxyl groups. Secondly, after 24 h, up to 25 modifications per 21 -mer RNA were observed in the mass spectrum (FIG. 4D). This excessive modification could possibly result from the excess Br-AIa-AI reagent reacting with the N-nucleobases under the water-free conditions.

[00108] To evaluate the hypothesis, the RNA21 after the 4 h or 24 h treatment with Br-AIa-AI was analyzed by UV-Vis spectroscopy, monitoring the shift of the peak at 260 nm (As®)), which corresponds to the absorbance of nucleobases (FIG. 4B). Interestingly, after 24 h of reaction in anhydrous DMSO, a significant shift (ca. 7 nm) of the nucleobase absorption peak and an increase in a peak at 310 nm (Auo) were observed, indicating an acylation reaction on the nitrogenous bases. After 4 h of reaction, the shape of the UV-vis spectrum remained largely similar to that of the untreated RNA21 (broken line in FIG. 4B). Reactions in 20% v/v DMSO for 4 h (FIG. 4B) did not induce such a peak shift, because under these conditions mainly 2'-6)-adducts were formed, It was hypothesized that at the beginning of a water-free acylation process, Br-AIa-AI preferentially reacts at the 2 -OFL since the amine groups in nucleobases are poorer nucleophiles than 2 -OH groups as a result of the electron delocalization. However, a higher number of coupling reactions to the amines on the nucleobases could be induced by extending the reaction time resulting in quantitative levels of initiator incorporation. A similar result was observed when DNA2I was used under anhydrous conditions. Despite the absence of 2 ^r -OHs, all of the DNA21 molecules reacted with up to 8 modifications as observed in a mass spectrum. In addition, a 6 nm shift of the peak in the absorption spectrum further supported the modifications on the DNA nucleobases. PAGE of RNA21 and DNA21 strands was performed. Retarded migration of RNA21 and DNA21 was observed after the incorporation of the ATRP initiators into the oligonucleotides. Thus the acylating reagents hereof may be extended to functionalization of polynucleotides generally via reaction with 2'-OH groups (when present) and/or with nitrogenous nucleobase. FIG. ID illustrates a generalized scheme for modification of polynucleotides via reaction with acylating reagents hereof whether the reaction occurs via 2 '-OH groups and/or nitrogenous nucleobases of the polynucleotide .

[00109] ATRP was performed with DNA21 or RNA21 initiators synthesized by the water-free approach. An increased monomer conversion of 65% for the RNA21 initiator (previously, 25% for the RNA21 initiator synthesized in 20% v/v DMSO in water, FIG. 2D) and 9% for the DNA21 initiator (previously, 0% in FIG. 2G) was observed by ^l H N’MR as a result of the increased number of initiators per RNA (and DNA) in anhydrous DMSO reactions. The SEC peak of the pOEOMA500-grafted DNA21, acylated in 100% DMSO, was observed in the high molecular weight region (elution volume of m 18.3 mL) as a result of polymer growth from the DNA. However, the polymerization product obtained from RNA21, synthesized in 100% DMSO, was too viscous to be injected to and analyzed by SEC. This is likely due to the accelerated termination reaction between inter- or intramolecular polymer chains induced by densely-packed initiating residues, which occurs as the conversion increases. The optimization of the polymerization conditions using sacrificial initiators may be used to solve the problem. The results of the polymerization from oligonucleotide initiators are summarized in the Table 2. Table 2. Summary of grafting from oligonucleotide in itiators.

Entry Oligonucleotide Acylation condition Initiator Dpr £ _onv « '^V ^S 'V ^S & per OUgO* ikL)A) ikDA)

1 RNA21 20% DMSO, 4 hr 6 924 25% 487 470 1.17

2 DNA21 20% DMSO, 4 hr 0 N/A 0% N/A N/A N/A

3 RNA21 ⁴ 20% DMSO, 4 hr 1.5 836 15% 307 425 1.13

4 RNA21 100% DMSO, 24 hr 23 N/A 65% 1.307 , ^lo ° N/A

VISCOUS

5 DNA21 100% DMSO. 24 hr 7 684 9% 186 350 1.15

³ The number of initiators per oligonucleotide was calculated from the corresponding mass spectrum. ‘The degree of polymerization (DP) was calculated using the following equation of the oligonucleotidepolymer conjugate - molar mass of oligonucleotide initiator) divided by 500, the molar mass of OEOMAw 'The conversion was determined by ‘H NMR (Figure S8). ‘^peDNA2l was introduced as the helper DNA prior to acylation.

[00110] Over the past decade, the potential of nucleic acids from biomass as a novel building block for the fabrication of sustainable biocompatible materials has been demonstrated. Further representative studies of the method hereof were performed to extend the scope of polynucleic acids that can be used in reactions hereof to a representati ve biomass RNA (ZWJRNA) extracted from torula yeast (FIG. 5A). Via reaction with nucleobases as described above, the reactions hereof may further be extended to biomass DNA (ZwtDNA). In a number of studies, ZVHRNA .macroinitiator was first prepared by mixing 10 mg of ZwzRNA with 100 pL of 0.6 M Br-Ala-Al reagent in anhydrous DMSO. After overnight incubation under anhydrous conditions with gentle shaking, almost complete dissolution of the ZwrRNA in DMSO was observed, indicating successful modifications (FIG. SB, right). When bmRNA was incubated in the DMSO without Br-Ala-AI (FIG. SB, left), the RNA remained mostly undissolved as a result of to the poor solubility of native RNA in organic solvents. To isolate the fonctionalized bmRNA (in the supernatant) and to remove the unreacted bmRNA (in a pellet form), the mixture was centrifuged and the supernatant was collected. To precipitate the bmRNA initiator from the supernatant, sodium acetate and isopropanol were added. The resulting precipitate containing the bmRNA initiator was collected by centrifugation, and if necessary, a 3K MWCO filter was used to further purify the sample. Finally, the bmRNA initiator was characterized by UV-Vis spectroscopy FIG. 5C). A change in the spectrum was observed after water-free functionalization (FIG. 5C), similar to the results obtained with RNA21 as a result of modifications to the nucleobases. The change in the shape of the UV-Vis spectrum of bmRNA (FIG. SC) after water-free acylation was less significant compared to the RNA oligomer (RNA21 , FIG. 4B). This observation may arise from the aggregation of the fouRNA strands in DMSO and the use of the higher amount of RN A substrate (15 gmoles and 0.42 pmoles of ribonucleotides for ZwzRNA and RNA21, respectively). Using ^l H NMR spectroscopy, the ratio of ribonucleotides to ATRP initiator residues incorporated into the bmRNA was estimated to be 4.25 acylations per 10 ribonucleotides.

[00111] Next, the capability of the &»RNA macroinitiator prepared by the water-free approach to initiate the photo-ATRP of OEOMAsw monomer was examined. As shown in Table 3, negligible monomer conversion was observed in negative control experiments lacking 2’-OH in the substrate (Table 3, entry 1 ), Br-Ala-Al treatment (Table 1 , entry 2), or greenlight for polymerization (Table 3, entry 3). In contrast, a high monomer conversion (>50%) was observed only when Br~A!a-AI -treated &nRNA was used as the macroinitiator under green light irradiation. In addition, the conversion gradually increased as more initiators were used for polymerization (Table 3. entries 4-7), which is consistent with other polymerization results in photo-ATRP. The polymerization products (bmRNA-pOEOMA) were isolated by using 100K MWCO filter and a Sep-Pak cartridge as mentioned above and then analyzed by SEC-MALS. The shift of the monomodal SEC-MALS traces in Fig. 50 demonstrated that the molecul ar weights of the polymer grafts from bwRNA were controlled by simply changing the concentration of the &nR’NA macroinitiator. SEC-MALS analysis of the Z>»?RNA macroinitiator showed a relatively low dispersity (£)) of 1.15 with a number average molecular weight (M,MAI.S) of 12.1 kDa (Table 3, entry 3). This observation may be a result of the lower number of modifications and the precipitation of short ZwRNA strands during the anhydrous acylation process .

Table 3. Results of grafting from biomass nucleic acids? . . . . Degree of

... . ... . ■ nucleic acid _b

Eutrv Initiator , , Conv. ... , f) polvmenzatio concentration (kDA) ¹ * _t n

1 Biomass DN A 0.5

2 ^li Biomass RNA 0.5 mg/mL 1% N/A N/A N/A ty??RN A

3 ^V Biomass RNA 0.5 mg/mL 1% 12.1 1.15 initiator

4 Biomass RNA 0.5 mg/mL 57% 1238.4 1.41 2452

5 Biomass RNA 1.5 mg/mL 60% 496.7 1.37 969

6 Biomass RNA 4.5 mg/mL 65% 239.6 1.22 455

7 Biomass RNA 9.0 mg/mL 68% 132.7 1.24 241

*SEC-MALS traces are shown in Figure 5D. Biomass DN A extracted from salmon was used as the initiator for entry i after Br-Afa*Al treatmart in anhydrous DMSO. Reaction conditions: [OEOMA$« ₍ ] ~ 300 mM, [EYHsj ~ 0.015 niM, I’CuBrs] ~ 0.9 mM, [TPMA] = 2.7 mM, [taRN A] ~ 0.5-9.0 mg/mL under the green LEDs (520 am, 3.7 tnW/oif ) for 30 min at r.t, in PBS. The conversion was determined by ^S H NMR spectroscopy (Figure S 13). '’The degree of polymerization for the chains grafted (rant fonRN A initiator was calculated using the following equation: (M>,wv,s of the polymer - MM-WS of NMRNA initiator (12.1 EDA, entry 3)) divided by 500. the molar mass of O.EOMA<«o. '7»»RN A, without treatment with Br-Ala-AI was used. -The reaction was performed in the dark, without green-light irradiation.

[00112] To determine the presence of RNA in &»RNA-pOEOMA conjugates, the polymerization products were stained with the RNA-staining fluorogenic dye (SYBR Gold). That cyanine-based dye exhibits high fluorescence enhancement upon binding to nucleic acids. SYBR Gold was used with untreated Z»??RNA, Zn??RNA macroinitiator, and ZbnRN 'A-pOEOMA hybrids from Table 3 dissol ved in water at a final concentration of 1 mg/mL, 100 nL of each sample was treated with SYBR Gold, and recorded the fluorescence intensity in a microplate reader (FIG. 5E). As expected, strong fluorescence enhancement by SYBR Gold was observed for all ZwRNA macroinitiators and &wRNA-pOEOMA hybrids. In contrast, no fluorescence was observed for pOEOMA initiated from a conventional ATRP initiator (i.e,, HEB1B, 2- hydroxyethyl a-bromoisobutyrate). Moreover, the lower fluorescence intensity of the ZwRNA macroinitiator or polymer-functionalized biomass RN A (FIG. 5E, entries 3-7) compared to the untreated d/nRNA was also observed, which may be a result of the inhibited intercalation of the SYBR Gold dye molecules between the planar nucleobases. [00113] An important feature of controlled radical polymerization is the living polymer chain end, which facilitates the synthesis of di- or multi-block copolymers. To investigate the fidelity of the chain end grafted from the ZwrRNA macroinitiator, a chain extension experiment was conducted (FIG. 5F). Initially, 6mRNA-pOEOMA was synthesized under the general polymerization conditions 146.7 kDa, conv. = 66%). An aliquot of the polymerization product was taken without further purification and combined with the ATRP catalytic mixture for the second polymerization. After 30 min of the second polymerization reaction under green light, 49% of monomer conversion was observed with a clear shifting of the SEC-MALS trace to the high molecular weight values indicating preserved chain end activity after the first polymerization (Table 4).

Table 4. Result of chain extension experiments from FIG. 5F.

Initiator ... ₃ MCMALS

Entry , . Conv. _r , . . i) eoncen (ration (kD A) 1.32 1.44 (From Entry 1 )

“Conversion was determined by *H NMR spectroscopy.

[00114] The functionalization of ZWJRNA and subsequent polymerization was applied to the fabrication of macroscopic structures. As shown in FIG. 6A, a hydrogel was synthesized by the photo-ATRP of OEOMA ₅₀₀ using the ZwzRNA macroinitiator in the presence of a crosslinker (PEGDMAiso, polyethylene glycol) dimethacrylate, average M. ~ 750). The polymerization reaction was performed in a 96-well plate using a photoreactor (520 nm). As a result of polymerization, a macroscopic hydrogel was obtained, as shown in FIG. 6B. When ZwRNA without Br-Ahi-Al treatment was used instead of b/nRNA macroinitiator, no polymerization occurred (FIG. 6B, right), indicating negligible polymerization initiation induced by the photredox/Cu dual catalysis.

[00115] Taking advantage of the increased solubility of ATRP initiator-functionalized RNA in DMSO. polymerization from the ZwRNA macroinitiator in an organic solvent (i.e,, DM SO) was pursued, which would provide a promising new approach for the fabrication of nucleic acid-polymer amphiphiles. In addition to water-soluble polymers, such as polyethylene glycol (PEG), that, for example, provide degradation resistance, other polymers such as poly(A«isopropylacrylamide) (pNIPAM), can provide temperature-responsiveness to RNA- based biomaterials. Using A-isopropylacrylamide (NIPAM) as the model monomer, polymerization reactions in 80% v/v DM SO in PBS were performed. The resulting ZMRNA- pNIPAM hybrid was dialyzed in ice-cold water and transferred to a 0.5-dram vial. As shown in FIG. 6C, when the temperature was increased above the lower critical solution temperature (LOST) of pNIPAM, the ^mRNA-pNlPAM hybrids aggregated and became a gel. In contrast, when 2?mRNA without Br-AIa-AI treatment was used instead of the ZwRNA macroinitiator, no polymerization of 'NIPAM was observed and the solution remained as a flowable liquid even after heating (FIG. 6C). Shrinkage of ZnuRNA-pNIPAM was also observed by dynamic light scattering. These results demonstrate that novel materials can be fabricated from fowRNA functionalized using ATRP initiator reagents hereof with a wide range of monomers under anhydrous polymerization conditions.

[00116] As described above, the simple crosslinking of biomass DNA constrains the possibility of engineering material properties since incorporating functional molecules (e.g., synthetic polymers) is challenging. To address this challenge, the acylation reagents hereof were extended, using the representative example of biomass RNA, to functionalize the RNA with polymerizable groups such as polymerizable vinyl groups (see, for example, FIG. 7 A, bottom). As described above in connection with the ATRP-initiator-ftmctionalized acyl imidazole reagents, this strategy for functionalization takes advantage of the 2'-hydroxyl groups in RNA. Functionalization with vinyl groups converts biomass RNA into acrylic crosslinkers which can undergo subsequent polymerization through radical polymerization. The biomass RNA-functionalization was leveraged through acyl imidazole chemistry as described above wherein the covalent reaction between 2*-hydroxyl groups in RNA with acylating reagents, particularly acyl imidazole, is used to form S’-O-adduct. The RN A can be modified with acyl imidazole reagents under the mild and biocompatible polymerization conditions. During the radical polymerization process, vinyl monomers can undergo copolymerization with two or more different types of monomers. As a result, a new material emerges with contributing properties from each monomer, leading to a tailored material for specific needs. Consequently, crosslinking methods hereof significantly broaden the scope of possible functionalities through radical (co)polymerization methods for biomass RNA-based- 3D structured materials. Such expansion opens the door to outstanding potential applicability in various fields, including, for example, 3D-printing, healthcare, electronics, and catalysis. [00117J As described above, acylating agents hereof may further be used to functionalize a polynuclear acid with one or more polymerizable groups. In a representative study, acrylamide-fimcfionalized acyl imidazole (AAm-AI) was synthesized as a model acylating reagent in reaction of 6-acrylamidohexanoic acid with 1,1 '-carbonyldiimidazole (GDI) in DMSO (FIG. 7A). The formation of the AAm-AI was confirmed by NMR spectroscopy. The freshly synthesized AAm-AI was added to commercially available biomass RNA extracted from torula yeast. The resulting acryiamido RNA crosslinker was isolated by precipitation with isopropanol. 2D heteronuclear single quantum coherence (HSQC) NMR analysis ( ¹ H- ¹³ C) of the biomass RN A before and after AAm-AI treatment confirmed that the acrylamide linkers were successfully incorporated in the biomass RNA . The results of a proton diffusion-ordered spectroscopy ( ¹ H-DOSY) study also showed that the protons in the RNA and the acryiamido moieties have identical diffusion coefficients, indicating that they were in the same molecule. The biomass RNA crosslinker was further characterized using size exclusion chromatography equipped with multi-angle light scattering (SEC-MALS) (A/».MALS = ^: 12 850, D 1.18).

[001181 The degree of AAm-AI modification of RNA was then quantified. It has been shown that the acylation yield can be influenced by the reaction conditions, including the amount of acylating reagent used or the ratio of cosolvent (e.g., DMSO) to water. Therefore, acryiamido RNA crosslinkers were synthesized under different acylation conditions (% DMSO v/y in water and the amount of AAm-AI), and the degree of acylation was determined using ^l H NMR. As shown in FIG. 7B, conducting RNA modification with a higher concentration of DMSO in water or using a larger amount of AAm-AI resulted in an increased number of acrylamide incorporation in the RNA. It should be noted that up to supemtoichiometric yield (12.8 acrylamide groups per 10 ribonucleotides) could be achieved when RNA was treated with 3 equivalents (3X) of AAm-AI compared to ribonucleotide in 100% DMSO overnight. The enhanced acylation under high % DMSO is attributed to the slower hydrolysis of AAm-AI under such conditions ( FIG. 7G). The RNA crossiinkers were further characterized by UV-Vis as shown in FIG. 7D. The shift of the characteristic absorption peak of RNA at 260 nm, corresponding to nucleobases. indicated that AAm-AI reacted with nitrogenous bases in addition to 2'~hydroxyl groups. The most significant shift (ca. 5 nm) was observed for the 3X, I 00% DMSO condition (FIG. 7D) compared to other conditions (ca. 0.5-2 tun) as water shows stronger nucleophilicity to most acylating reagents than amine groups on RNA bases. These results indicate that tire binding site and the number of acryiamido functional groups on the RNA crosslinker can be engineered by tuning the experimental conditions, including, for example, the acylation media or the amount of acylating reagent.

[00119] With the acrylamido RNA crosslinkers in hand, the capability of acrylamido residues in RNA to undergo polymerization via free radical polymerization (FRP) was tested. A summary of the reaction conditions for the synthesis of the hydrogels is shown in Table 5. Three different RNA crosslinkers were synthesized in 100%, 50%, or 25% DMSO in water (v/v) using 3X of AAm-AI to ribonucleotide, respectively. Each RNA crosslinkers was then homopolymerized by FRP using ammonium persulfate (APS) and tetramethylefhylenediamine (TEMED) in water. After 5 min of polymerization at room temperature, the RNA hydrogels were stained with GelRed, a nucleic acid-intercalating dye, for visualization (FIG. 8, left-top). Notably, the RNA hydrogel (25R _{1 00} ), fabricated using the RNA crosslinker prepared in 25% DMSO (v/y), could not free-stand independently outside of water under the polymerization conditions tested. This observation may be attributable to the lower number of crosslinking points compared to RNA crosslinkers synthesized in 100% (lOORm) or 50% (50.Rm) DMSO ( v/t'7 (FIG. 78).

Table 5. Summary of the reaction conditions for the synthesis of hydrogels in FIG. 8.

Acylation Volume

Entr RNA mass

Name condilimis , _t . v omonomer (RNA

(wt%r cone.)

25 iiL FRP

100% v v 3.75 mg

1 lOORt® (150 (APS

25 uL FRP

50% i7v 3.75

2 5ORmo ( 150 (APS.

DMSO (100 w mg/mL) TEMED)

25 HL FRP

25% Wv 3.75 mg

3 25Rw (150 (APS,

DMSO (100 wt%) mg/m.L) TEMED)

N equivalents of AAm-AI compared to ribonucleotides were used, “The weight percentage (wt%) of RNA in the gel was calculated from the following equation: (mass of RN A in the gel) / (mass of RN A mass of comonomer) X 100. "Reaction was performed in water.

[00120] Chemical modifications of nucleic acids can increase the resistance of nucleic acids to enzymatic degradation by introducing structural alterations and steric hindrance. In view of this, it was hypothesized that the different degrees of acylation would result in distinct degradation behaviors of RNA hydrogels. To test that hypothesis, the biodegradability of the three RNA hydrogel variants (100R ₁₀₀ , 50 R _{1 00} , and 25R _{1 00} ) under biologically relevant conditions (i.e., 15% fetal bovine serum, FBS) were examined as shown in FIG. 8, Rapid and nearly complete degradation of 25Rm, and a relatively stow degradation of 50Rm was observed, which was evidenced by the diffusion of GelRed to the supernatant. While 25R _{1 00} and 50Rm were completely degraded within 24 h, 100R ₁₀₀ remained nearly intact until 18 days of inc uba tion, The complete degradation of 100Rm was finally observed after 30 days of incubation. These results indicated that the stability and durability of the RN A hydrogel can be engineered by selecting an appropriate RNA crosslinker, which can, for example, be useful for the controlled release of cargo loaded in the hydrogel.

[00121] To demonstrate the versatility of the methodology hereof the copolymerization of the RNA crosslinker with various acrylic monomers (FIG. 9A) was investigated. RNA crosslinkers synthesized under different concentrations of DMSO (25, 50, and 100% v/v) were copolymerized with A-isopropylacndamide (NIP AM) to make RNA-NIPAM hybrid gels (2-4 in FIG. 98). To facilitate abbreviated nomenclature, the RNA hybrid gels are referred to as xR _y Comonomer _z . where x is the % DMSO (Wv) used in the acylation process, ‘Comonomer* represents the abbreviation of the comonomer ( for example, NIPAM), and y and z indicate the weight percentages of RNA and the comonomer in the gel, respectively. After the successful copolymerization of RNA crosslinkers with NIPAM, the hydrogels were stained with GelRed (FIG. 9B) by soaking the hydrogels in 20-100X GelRed in water. After overnight incubation under gentle shaking, all three RNA-NIPAM hybrid gels were stained by GelRed, confirming the presence of RNA in the hydrogels. Interestingly, swelling of 25R55NIPAM45 (2 in FIG. 98) and 50RssNIPAM45 (3 in FIG. .98) was observed, indicating a lower degree of crosslinking caused by the lower acrylamide content in the RNA crosslinkers synthesized in 25% and 50% DMSO (v/v). NIPAM was also copolymerized with acrylamide/bisacrylamide 29:1 mix (AAmlx) at a final concentration of 5% to make NIPAM gel without RNA (1 in FIG. 98), Despite the subsequent staining with GelRed dye, the NIPAM gel remained unstained as a result of the absence of RNA. The degradation of the RNA-NIPAM copolymeric hydrogel in 15% FBS was also examined (Table 6). The copolymeric hybrid RNA gels exhibited a retarded degradation, possibly attributed to a reduced RNA content within the gel and steric hindrance. Table 6. Summary of the reaction conditions for the synthesis of hydrogels.

Acvla tion , , V ohi me

Entr * RNA mass

*3 equivalents of AAm-Al compared to ribonucleotides were used, ^! The weight percentage (wt%) of RNA in the gel was calculated from the following equation: (mass of RNA in the gel) / (mass of RNA * mass of comonomer) X 1(K), “Reaction was perfmmed in water.

[00122] A reaction was also carried out in a custom-designed mold by copolyrnerizing RNA crosslinker using the AAmix as the diluent (FIG. 9C). The mold was charged with RNA crosslinker synthesized in 25% DMSO (v/v) and AAmix (final concentration of 2%) followed by the addition of APS and TEMED to initiate FRP. After 5 min of incubation, the RNA- acrylamide hybrid gel (ISR^AAmixjj) was demolded. The resulting copolymeric RNA hydrogels exhibited solid structural integrity, enabling them to tree-stand and maintain their shape in the air as a result of successful copolymerization with AAmix,

[00123] To quantify the RNA crosslinker-assisted enhancement of stiffness, cylindrical RNA-acrylamide hybrid gels were fabricated and mechanical property tests conducted in the compression mode (FIG. 9D). RN A crosslinkers synthesized in 25% or 100% DMSO (v/v) were copolymerized with 8% AAmix at the final concentrations of 10, 30, and 90 mg/mL, respectively. FIG. 9D demonstrates that increasing the amount of RNA crosslinker in RNA- AAmix hybrids led to higher compression moduli of the copolymeric hydrogels, indicating that the addition of RN A crosslinker reinforced the mechanical properties of the hybrid gels. The use of RNA crosslinker synthesized in a higher concentration of DMSO facilitated the formation of more strongly crosslinked networks, consistent with previous findings. In contrast, the addition of unmodified biomass RNA (R11AAmix89) showed no significant mprovement in the mechanical properties. [00124] In addition to NIP AM and acrylamides, the capability to copolymerize hydrophobic monomers with the RNA crosslinker offers the potential to engineer a wide range of material properties, including enhanced stability in organic solvents and hydrophobic characteristics. The high solubility of the RNA crosslinker in DM SO (>200 mg/mL) prompted a study of the copolymerization of the RNA crosslinker with hydrophobic monomers in organic solvent (that is, DMSO). As shown in FIG. 9E, the RNA crosslinker was mixed with methyl acrylate (MA) and copolymerized in DMSO by photoinduced FRP (X ~ 365 nm) using Irgacure 2959 as the initiator. After 30 min of polymerization under UV light the RNA-MA hybrid material, IOORJTMASS (7 in FIG. 9E), was successfully fabricated. In contrast, 5 (MA with Irgacure 2959) or 6 (MA and unmodified RNA with Irgacure 2959) in FIG. 9E did not form a gel, indicating the successful formation of a polymeric network assisted by the RNA crosslinker. In addition to MA, a variety of other acrylic monomers with distinct hydrophobicity were also successfully copolymerized with the RNA crosslinker and characterized by Raman spectroscopy and thermogravimetry to confinn the identity of the copolymeric material. As shown in FIG. 9F, contact angle measurements of RNA hydrogel (100R _{1 00} ) aud copolymeric RNA hybrid materials made with dimethyl acrylamide (100R29D MAAm71) or MA (IOORJJMA®*) were also conducted. The distinct contact angles of the hydrogels indicate that the copolymerization of appropriate acrylic monomers with the RNA crosslinker could tune the hydrophobicity of the RNA hydrogels.

[00125] Summaries of the reaction conditions for the hydrogel synthesis in water

FIG. 9B-9D) and in DMSO (FIG. 9E and 9F) are shown in Tables 7 and 8, respectively.

Table 7. Summary of the reaction conditions for the synthesis of hydrogels shown in FIGS. 4B-4D.

„ . . . . . . Volume

Entr Acylation RN A mass ,

*3 equivalents of AAm-Al compared to ribonucleotides were used. ^b The weight percentage (wt%) of RNA is the gel was calculated from the following equation: (mass of RNA in the gel) Z (mass of RNA -t mass of comonomer) X 100. -'Reaction was performed in water. ^d 5% acrylamide mix was added io 1.06 mg of NIPAM as the crosslinker. "Biomass RNA without the treatment of AAm-Al was used. Table 8. Summary of the synthetic conditions for the hydrogels. mg'ml..) TEMFD)

*3 equivalents of AAm-AI compared to ribonucteoiides were used. '’The weight percentage Cwi%) of RNA in the gel was calculated from the following equation: (mass of RNA in die gel) / (mass of RNA + mass of comonomer) X 100, 'Reaction was performed in DMSO under the irradiation ofUV light (1 - 365 nrn). ^Biomass RN A without the treatment of AAm-AI was used. 'Reaction was performed in water. Abbreviations: DMAAm (dimethyl acrylamide), HEMA (hydroxyethyl methacrylate ), BA (butyl acrylate), MA (methyl acrylate), and AN (acrylonitrile).

[00126| Reversible deactivation radical polymerization (RDRP) methods are changing the world by enabling the precise synthesis of polymers and materials in a property-controlled manner. Atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization are among the most widely used RDRP techniques. enabling precise control over the properties of synthetic polymers, including molecular weights, molecular weight distributions, composition, and architectures (e.g., multi-block copolymers, star-shaped polymers, etc.). As a result,, polymeric networks and multi-scale materials made by ATRP and RAFT have found various applications in electronics, adhesives, coatings, lubricants, and healthcare.

[00127] The possibility of polymerizing the RNA cross tinker through ATR.P and RAFT, which could provide an alternati ve route to engineer the material properties, in addition to using different comonomers was thus studied. To ensure well-controlled polymerization via ATRP and RAFT, another acylating reagent was designed with methacrylate functionalities using hydroxyethyl methacrylate (HEMA) and CD1. The resulting HEM A- functionalized imidazole carbamate (HEMA-CM) was treated to biomass RNA in 50% DM SO in water (v/y) overnight. Characterization of the methacrylic RNA crosslinker product using UV-Vis and ’H NMR spectroscopy showed slightly reduced functionalization efficiency of HEMA-CM (3.6 methacrylate groups per 10 ribonucleotides ) compared to AAm-Al (5.5 acrylamide groups per 10 ribonucleotides, FIG, 7B).

[00128] The methacrylic RNA crosslinker was then polymerized by photo- ATRP or photoinduced electron/energy transfer RAFT polymerization (PET-RAFT) in PBS under green light irradiation using eosin Y (EYHs) and oligofethylene oxide) methyl ether methacrylate (OEOMA ₅₀₀ , M = 500) as the photocatalyst and the model comonomer, respectively (FIG. 10A). See Szczepaniak, G.; Jeong, J.; Kapil, K.; Dadashi-Silab, S.; Yemeni, S. S.; Ratajczyk, P.; LathwaL S.; Schild, D. J.; Das, S. R.; Matyjaszewski, K., Open-air green-light- driven ATRP enabled by dual photoredox/copper catalysis. Chem. Sei. 2022, 13 (39), 11540- 1 1550., Jeong, I; Szczepaniak, G.; Das, S. R.; Matvjasz.ewski, K.., Synthesis of RNA- Amphiphiles via Atom Transfer Radical Polymerization in the Organic Phase. Pmvs. Chem, 2023, 1 (5), 326-331 , Jeong, J,; Szczepaniak, G.; Das, S. R.; Matyjaszewski, K., Expanding the architectural horizon of nucleic-acid-polymer biohybrids by site-controlled incorporation of ATRP initiators in DNA and RNA. Chem 2023, Phommalysack-Lovan, 1; Chu, Y.; Boyer, G.; Xu, J., PET-RAFT polymerisation: towards green and precision polymer manufacturing. Chem. Commun. 2018, 54 (50), 6591-6606, Xu, J.; Shanmugam, S.; Duong, H, T.; Boyer, C., Organo-photocatalysts for photoinduced electron transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization. Pofym. Chem. 2015, 6 (31), 5615-5624, and Zhang, T,; Wu, Z.; Ng, G,; Boyer, C. A. J. M., Design of oxygen-tolerant Photo-RAFT system for protein-polymer conjugation achieving high bioactivity. Angew. Chem., hit. Ed. 2(123, e202309582, the disclosure of which are incorporated herein by refereiic. A notable photobleaching of eosin Y was observed after 30 min of PET-RAFT polymerization (FIG. 10B), consistent with previous findings. In contrast, the RNA hydrogel made by EY7Cu- mediated photo- ATRP retained its pink color, originating from the EYHs dye (FIG. 10C). This observation is due to the rapid electron transfer from excited eosin Y in the triplet state to the [Cu ⁿ /L]2 ⁺ complex. Next, the swelling ratio of the three hydrogels made by PET-RAFT, photo- ATRP, and FRP was measured to compare the effect of the polymerization method on the network (FIG. 10D), A significant difference in the swelling behavior was observed among the RNA-QEOMASCM) hybrid networks prepared by PET-RAFT, photo- ATRP, and FRP. The distinct swelling ratios are likely due to the use of multi-handle crosslinkers and the difference in the initiation efficiencies of each photopolymerization method. The rapid initiation of the photo-ATRP process, facilitated by the efficient electron transfer to the [Cull/L] 2 ⁺ complex, led to a simultaneous and accelerated polymerization process, leading to a more efficient crosslinking, smaller network mesh size, and lower swelling ratio. A summary of the reaction conditions for the synthesis of the hydrogels is shown in Table 9.

Table 9. Summary of the reaction conditions for the synthesis of hydrogels in FIG. 10.

”6 equivalents of HEMA-CM compared to ribonucleotides were used. ^{ The weight percentage (wt%) of RNA in die gel was calculated front the following equation: (mass of RNA tn {lie gel) / (mass of RNA t- mass of comonomer) X 100. Reaction was performed in IX PBS under the irradiation of green light (X » 540 m, for PET-RAFT or EY-ATRP) or UV light (X - 365 nm, for FRP), respectively.

[00129] Nucleic acids interact with transition metal ions such as Au, Ag, Pt, and Co through diverse mechanisms, including coordination, intercalation, and electrostatic interaction. The resulting metalized nucleic acids possess several applications, such as electrochemiluminescence probes, catalysis, and nanopatteming. It was hypothesized that the metallization of RNA (for example, coordination of metal ions to nucleobases and the subsequent reduction of metal by electron-rich nucleobases) could also occur on the polymeric RNA network (FIG. 11 A), as previously observed in the DNA-based materials. To test the hypothesis, RNA-acrylamide copolymeric gels (50R§oAAmlxso) were synthesized by FRP and washed the gel by soaking it in water overnight under gentle shaking. The gel was then treated with 400 fflM AgNCh (3 in FIG. I IB), 100 mM AgNOs (2 in FIG. 11 B), or water (1 in FIG. 11B), respectively. The shrinking of 50Rs#AAmixsf> was noticed after the 3 h of AgNO? treatments, which may be attributed to the crosslinking induced by the formation of Cytosine- Ag ^: -Cytosme bridges. In contrast, the treatment of AgNOs with the polyacrylamide gels did not result in a noticeable shrinking of the gel (4—6 in Figure 6B), indicating the RNA-selective coordination of Ag\ As shown in FIG. 11 C, the AgNO .-treated RNA gel (2 in FIG. 11 B) and the AgNO3-treated polyacrylamide gel (5 in FIG. FI B) were further characterized using a scanning electron microscope (SEM). The cross-sectional energy dispersive X-ray (EDX) analysis of 2 (FIG. .1 IC. top lane) and 5 (FIG. 11C, bottom lane) confirmed the RNA-selective binding of Agf Elemental analysis showed that treatment with a higher concentration of AgNOa may result in a different loading of Ag in the hydrogel.

[00130] By harnessing the metal doping onto the RNA hydrogel, various applications ranging from diagnostics, catalysis, and electronic devices may be achieved. As an example, measurements to assess the conductivity of RNA-acrylamide hybrid gels (FIG. UD) were conducted. Different RNA contents (0 -50 wt%) were used to synthesize these gels and they were incubated in 100 mM AgNOa overnight. The successful binding of Ag" to RNA and crosslinking was evidenced by the shrinking of the RNA hydrogels (FIG. 11D). The dried hydrogels were placed between two stainless steel plates in CR2032-type coin cells for the conductivity comparison. As demonstrated in the Current- Voltage (IV) measurement results in FIG. 110, the AgNOs treatment played an important role in inducing the conducti vity of the RNA hydrogel. Moreover, stronger conductivity were observed when more RNA was used in the hydrogel fabrication. Those results indicate that the treatment of metal precursor solutions may provide an additional possibility to engineer hydrogel properties in a post-synthetic manner. A summary of the reaction conditions for the synthesis of the hydrogels is shown in Table 11.

Table 1.1. Summary of the reaction conditions for the synthesis of hydrogels in FIG. 11.

(Figure AD) DMSO (33 v\t%) (40 mg 'niL) (ov ernight)

Polx aery lanude

220 pl

14 gel

(N Ai (overnight) d unite nD)

All cds were sv nilu-si/ed fix free sadicisl poly mes s/.tison m w.iur usine AFS ,md I'E MtO using :sc ^; y Dmi<.(c m=v

(filial concentration of 8%) as tlie (oojmononier. ^,! 3 equivalents of AAin-A.l compared to ribonucleotides were used. ’Tlie weight percentage (wt%) of RNA in the gel was calculated from the following equation: (mass of RNA in (lie gel) / (mass of RNA + mass of comonomer).

[001311 In summary, acylating reagents such as acyl imidazole reagents including a conjugated RDRP (for example.. ATR.P) initiator, a conjugated chain transfer agent for RDRP (for example, RAFF) polymerization, or a conjugated polymerizable reagent may be used to functionalize polynucleic acids/poly nucleotides (including, for example, DNA and RNA) for further reaction. The scope of polynucleic substrates extend from short synthetic oligonucleotides to sequences extracted from biomass.

[00132] The versatile approach to polynucleic acid-polymer hybrids, and, particularly, RNA-polymer hybrids, hereof circumvents challenges of other coupling methods, including azide-alkyne cycloadditions, electrostatic interactions, or hydrogen bonding. The direct, and universal incorporation of acyl reagent-based RDRP initiators or chain transfer agents for RDRP and subsequent polymerization will, tor example, expand the scope of RNA. substrates for the combination with polymers and increase their utility as innovative biomaterials. In addition, the high coupling yield, and thereby improved solubility of RNA initiators in the organic-phase, will significantly broaden the choice of polymerizable monomers (for example, for used nucleic acid therapeutics or pathogen detection). The methodologies and cornpositions/reagents hereof are readily applicable to both post-synthetic and nature-derived RN A and DNA and can be used to engineer the properties of a variety of RNA-based or DNA- based macromolecular hybrids and assemblies providing access to a wide variety of RNA- polymer or DNA-polymer hybrids.

[00133] Further, using acylation reagents hereof functionalized with polymerizable agents handles, the first successful conversion of biomass RNA into an acrylic monomer/ci'osslinker was achieved. The resulting acrylamide RNA crosslinker could undergo radical polymerization with a diverse range of other acrylic monomers. Studies hereof demonstrated the polymerization of the methacrylic RNA monomericrosslinker via RDRP techniques (for example, photo-ATRP and PET-RAFT), enabling the lubrication of copolymeric RNA gels with customizable swelling ratios through the choice of the polymerization method. The metallization of RNA with silver ions demonstrated its potential to enhance the electrical conductivity of biomass RNA-based materials hereof. The method and compositions hereof provide new possibilities for biomass RNA-based material fabrication with tailored properties, while overcoming the challenges associated with conventional synthetic strategies. The methods hereof may further accelerate advancements in the emerging field of biomass nucleic acid-based materials, enabling diverse applications such as drug release, electronics, and catalysis. [00134] Experimental

[00135] Materials. Unless otherwise stated, all the chemicals, including torula yeast RNA (type VI) were purchased from Sigma Aldrich. Molecular weight cut-off (MWCO) filters (Amicon ultra centrifugal filter) were purchased from Sigma Aldrich. All the oligonucleotides used in this study were purchased from IDT. Tris(2>pyridylmethyl)am.ine (TPMA) was purchased from AmBeed. Carbonyldiimidazole (GDI) was purchased from TCI America. All the organic solvents. 40% PAGE gel mix, 10X phosphate-buffered saline (PBS ), and I OX tris- borate-EDTA buffer were purchased from Fisher Scientific. SYBR Gold dye (10000X in DMSO) was purchased from Invitrogen. Sep~Pak Cl 8 cartridge was purchased from Waters. DNase I was purchased from New England BioLabs. 96 well plate was purchased from Greiner (CellStar ®). Me<>TREN trisi2-(dimethylainino)ethyl]amine) was received from Koei Chemical Co., Ltd.

[00136] Instruments. ^l H NJMR spectra were recorded by Broker Avarice III 500 MHz spectrometer NanoDrop One UV-Vis spectrophotometer (ThermoFisher Scientific) was used to obtain the absorbance spectrum. Polyacrylamide gel after PAGE was imaged by Typhoon FLA 9000 gel scanner (GE Healthcare Life Sciences). UltrafleXtreme MALDI-TOF Mass Spectrometer (Broker) was used for the characterization of oligonucleotides using MTP 384 Target Plate Ground Steel (Broker). The fluorescence intensity of SYBR Gold was measured by using Infinite® Ml 000 (Tec-an) microplate reader. The green LED strip (X = ^: 520 nm) for polymerization was purchased from aspect LED and the strip was mounted inside a glass jar (height = 7 cm, diameter = ^: 9 cm). Fabrication of biomass RNA gel was carried out using Lumidox II 96-Well LED Arrays (X ~ 540 nm) and Lumidox II Controller. Zetasizer Nano ZS (Malvern) was used for dynamic light scattering analysis. UV-VIS spectrometer (Lambda 2, PerkinElmer) was used for the determination of LCST of RNA-pNIPAM conjugate. of Br-Ala-AI, reagent. p-Alanine initiator (Br-Ala, A-a- bromoisobutyiyl-p-aianine) was synthesized by following the previously reported procedure.’ Next, 1.2 M CDI stock and 1.2 M Br-AIa initiator were prepared in the separate Eppendorf tube by dissolving 194,58 mg of GDI and 285.7 mg of Br-Aia in anhydrous DMSO at the final volume of 1 mL, respectively. Finally, equal volumes of 1.2 M CDI stock and 1.2 M Br~AIa were mixed and incubated for 10 min at room temperature under gentle shaking. Br-AIa- functionalized acy l imidazole reagent (Br-Ala-AI) at 0.6 M concentration in anhydrous DMSO was stored in a — 20°C freezer until further use. [00138] General procedure for oligonucleotide fanctionaliza tion with Br- Ala-AL 30 nmol of oligonucleotide substrate and 150 pL of 0.6 M Br-Ala-AI were mixed and the volume was brought to 750 p.L by adding water (final DMSO concentration is 20% v/v). The mixture was thoroughly mixed, followed by incubation for 4 h at room temperature under gentle shaking. Next, functionalized oligonucleotide was precipitated by using sodium acetate and 1.5X volume of isopropanol at -80 G for overnight. The precipitates were collected by centrifugation (13000g, 30 min) at 4X2. The pellet was redissolved in 500 pL of nuclease- free water. Then, the solution was further purified by using 3K MWCO filter (Amicoti ultra centrifugal filter) for three repeating centrifugations. After the purification step using the MWCO filter, the solution remaining in the filter was collected and the concentration of oligonucleotide was determined by measuring Aswi and the appropriate extinction coefficient calculated by Oligo Analyzer (IDT). Mass spectra of the oligonucleotides were recorded by using 3-Hydroxypicoliaic acid dissolved in 50% acetonitrile in water containing 10 mg/mL diammonium hydrogen citrate as a matrix for MALDI-TOF.

[00139] General procedure for EY/Cu-catalyzed photo-ATRP. Prior to polymerization, stock solutions of reagents were prepared as follows:

CuBn stock : 15 mg of CuBrs in 1 194 pL of 50% w'v DMSO in water

TPMA stock : 15 mg of TPM A in 153.6 pL of DMSO

EYHs stock : 9.72 mg of EYH ₂ in 10 mL of DMSO

OEOMA ₅₀₀ stock : 250 mg of OEOMA ₅₀₀ in 1 mL of water

[00140] Next, 150 pL of OEOMA ₅₀₀ stock, 4 pl. ofCuBn stock, 2 uL of TPMA stock, 2.5 pl. of EYH? stock. 25 pL of 10X PBS and oligonucleotide initiator (final concentration of 75 pM) was mixed, and the final volume of the mixture was brought to 250 pL by adding water. The reaction cocktail was transferred into a 250 uL glass insert followed by irradiation of green light (X ~ 520 nm, 3.7 mW cm" ² ) at room temperature to initiate polymerization. The final concentrations of reagents were as follows: [OEOMA ₅₀₀ ] = ^: 300 mM, [CuBn] = ^: 0.9 mM, [TPMA] ~ 2.7 mM, and [EYHa] - 0.015 mM. After 30 mln of polymerization under green light, 50 pL of the reaction mixture was used for ^! H NMR analysis to determine monomer conversion. The rest of the mixture was used for the determination of absolute molecular weight (Afsi.MALs) using size-exclusion chromatography equipped with a multi-angle light scatering detector (SEC-MALS). PBS was used as an eluent for SEC-MALS.

[00141] Controlled initiator incorporation in RNA with helper DNA. 20 nmol of RNA substrate and 32 nmol of corresponding helper DNA were mixed in a hybridization buffer ( 150 mM NaCI, 100 mM MOPS, 10 mM MgCb). The mixture was annealed by heating at 95 C for 8 min in a heat block and slowly cooling down to room temperature at the rate of approximately 0.5‘CZmin. Following the annealing process, 0.6 M Br-Ala-AI (final concentration of 0.12 Mj and 0.5 M MOPS buffer (pH 7.5) containing 50 mM MgCh and 0,5 M Nad was added at the final concentration of 100 mM of MOPS buffer. The mixture was incubated for 4 h at room temperature under gentle shaking. To the incubated mixture was added 5 pL of DNase I (10 units) and 12 pL of 10X DNase 1 reaction buffer (100 mM Tris-HCI, 25 mM MgCh, and 5 mM CaCb. pH 7.6). The mixture was incubated at 37 C for I h. After the incubation, the functionalized RNA was purified by using 3K MWCO filters as presented above and the concentration of RNA was calculated by measuring A260 and using Beer-Lambert Law.

[00142] Procedure for the water-free RN A functionalization. 100 pL of 0.6 M Br-Ala- AI in DM SO were mixed with 20 nmol of lyophilized RNA pellet or 10 mg of biomass RNA (car. 15 pinoles). After 24 hours of incubation at room temperature, functionalized RNA strands were purified by isopropanol precipitation and subsequent dialysis using MWCO filters as presented above. Finally, the absorbance maximum of the nucleobases in the range of 250-260 nm was measured to calculate the concentration of RNA using Beer-Lambert Law.

[00143] Chain extension experiment using biomass RNA macroinitiator. 150 gL of OEOMAw stock, 4 gL ofCuBrs stock, 2 pL of TPMA stock, 2.5 pL of EYH2 stock, 25 pL of 10X PBS and ZwRNA macroinitiator (final concentration of 6 mg/mL) was mixed, and the final volume of the mixture was brought to 250 pL by adding water. The ATRP “cocktail” was transferred into a 250 gL glass followed by irradiation of green light (1 ~ 520 nm, 3.7 mW cm' ² ) at room temperature to initiate polymerization. After 30 min of polymerization under green light, 125 p.L of the reaction mixture was used for analysis using ^! H NMR spectroscopy (to determine monomer conversion, conversion j st) and SEC-MALS. To the rest of the mixture was added 75 gl, of OEOMA500 stock, 2 p.L of CuBn stock, 1 pL of TPMA stock, 1.25 pl., of EYH2 stock, 12.5 pL of 10X PBS and 30.4 ph of water. The mixture was thoroughly mixed and transferred into a 250 gL glass followed by irradiation of green light to restart polymerization. After 30 min of reaction, 50 pL of the mixture was used for ^{ H NMR analysis to determine monomer conversion (conversions®}). The rest of the mixture was used for SEC- MALS analysis. The actual monomer conversion of the 2 ^nd polymerization (conversions^®^) was determined by the equation presented below.

[00144] faocgfaire for the ..fabrication of hffjRNA-p(>EOM A .hydrogeLhi %,weH plate.

Prior to polymerization, PEGDMAtso stock was prepared by dissolving 450 mg of PEGDMA.750 in 1 mt of water. Next, 150 pL of OEOMA ₅₀₀ stock, 4 uL of CuBn stock, 2 pL of TPMA stock, 2.5 pL of EYEls stock, 25 pL of 10X PBS. 25 pL of PEGDMA?® stock and ZwzRNA. macroinitiator (final concentration of 0.5 tng/mL) was mixed, and the final volume of the mixture was brought to 250 pL by adding water. The ATRP “cocktail” was transferred into a 96-w'ell plate followed by irradiation of green light (X ~ 540 nm, 20 mW at room temperature. After 30 min of polymerization, the LED light was turned off to stop the reaction.

[00145] Grafting N I PAM from bwRN A macrpinitiator. Prior to polymerization. N IP AM stock was prepared by dissolving 339.5 mg of NIP AM in DMSO at a final volume of 1 mL. Next, 83 pL of N1PAM stock, 12 pL of CuBn stock, 6 pL of TPMA stock, 7.5 pL of EYHs stock. 10 pL of DMF, 25 pL of water, 25 pL of 10X PBS and (wzRN A macroinitiator stock in DMSO was mixed (final concentration of 0.5 mg/mh), and the final volume of the mixture was brought to 250 pL by adding DMSO. The ATRP “cocktail” was transferred into a 250 pL glass insert followed by irradiation of green light (1 = 520 nm, 3.7 mW cm* ² ) at room temperature to ini tiate polymerization. The final concemrat.ions of reagents were as follows: [NIP AM] = 1000 mM, [CuBn] = 2.7 mM, [TPMA] = 8.1 mM, and [EYHs] = 0.045 mM. After 30 min of polymerization under green light, polymerization was stopped by turning the light off.

[00146] Genera] procedure for the synthesis of A Am-AI, 161.2 mg (1 mmol) of CD1 was dissolved in 650 pL of DMSO. To the dissolved CD1 in DMSO, 185.2 mg (1 mmol) of 6- acrylamidohexaaoic acid was added and the final volume was brought to 1 mL by the addition of DMSO. The resulting A Am- Al stock (1 M) was incubated at room temperature for 30 min under gentle shaking.

[00147] General procedure for the synthesis of the acrylamido RNA crosslinkers. 40 mg of yeast RNA was mixed with 400 pL of I M AAm-AI stock. For the acylation under the 25% or 50% v/v DMSO in water, an additional 1200 pL or 400 pL of nuclease-free water was added, respectively. Alter the overnight incubation at room temperature under gentle shaking, the resulting RNA crosslinker was precipitated by the addition of 3M sodium acetate (1/10 volume) and isopropanol (1 .5 volume). The precipitated RNA crosslinker was isolated by centrifugation (13000 rpm, 15 min) at 4 °C The isolated RNA crosslinker pellet was redissolved in water and further purified by additional precipitation and centrifugation. Finally, the purified RNA crosslinker pellet was dissolved in water and the concentration of RNA (mg/mL) was determined by measuring A260 (extinction coefficient - 40 (pg/mL)'' ^! cm' ^! ). To estimate of the degree of acylation, ca. 3 mg of RNA crosslinker was dissolved in 600 uL of DsO followed by ¹ H NMR analysis.

[00148] General procedure for the fabrication of RNA hydrogels via FRP in water. T o hcimopolynierize the RNA crosslinker in a reaction volume of 50 gL, acrylamido RNA crosslinker (final concentration of 150 -250 mg/mL) was taken and the volume was adjusted to 44 gL by adding water. Subsequently, 1 pL of TEMED and 5 pL of 10% APS in water were added and thoroughly mixed. The resulting mixture was incubated at room temperature for 3 min to polymerize. For the copolymerization of the RNA. crosslinker, 3 M NIPAM stock (339.5 mg in 1 mt DMSO) or 40% acrylamide mix (actylamideibisacrylamide ~ 29: 1 ) was prepared. Next, acrylamido RNA crosslinker (final concentration of 5-90 mg/mL) was mixed with NIPAM stock (final concentration of 250 mM) or acrylamide mix (final concentration of 2- 8%) and the volume was brought to 44 gL by adding water. Subsequently, 1 g.L of TEMED and 5 pL of 10% APS in water were added and thoroughly mixed. The resulting mixture was incubated at room temperature for 3 min to polymerize.

[00149] The foregoing description and accompanying drawings set forth a number of representative embodiments at the present time. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light, of the foregoing teachings without departing from the scope hereof, which is indicated by the following claims rather than by the foregoing description. All changes and variations that fall within the meaning and range of equivalency of the claims are to be embraced within their scope.